[Link].

com/scientificreports

OPEN New insights in Microbial Fuel Cells:

For the development of long lasting portable microbial fuel cells (MFCs) new strategies are necessary
received: 17 March 2016
to overcome critical issues such as hydraulic pump system and the biochemical substrate retrieval
accepted: 14 June 2016 overtime to sustain bacteria metabolism. The present work proposes the use of a synthetic solid anolyte
Published: 04 July 2016 (SSA), constituted by agar, carbonaceous and nitrogen sources dissolved into diluted seawater. Results
of a month-test showed the potential of the new SSA-MFC as a long lasting low energy consuming
system.

Renewable energy sources (RES) are important contributors to the transition to a low-carbon society, being able
to mitigate emissions of greenhouse gases, lower other environmental pressures associated with conventional
energy production and overturn the dominance of fossil fuels1. Among the several RES, developed and under
development, microbial fuel cells (MFCs) have emerged, during the last decade, as one of the most versatile
and promising one. A key reason that could make the MFCs a strategic technology is related to their working
principle: they can generate electricity through the catalytic activity of exoelectrogenic bacteria involved in the
anaerobic oxidation of organic substrates acting as low grade fuels2–4. The amount of energy that can be obtained
by MFCs is relatively low if compared to other fuel cell technologies2, but they have a unique feature in the field
of fuel cells: they can harvest chemical energy from several classes of wastes, with the potential to effectively
and directly convert into electrical energy several non-purified organic substrates, naturally present in different
environments. Moreover, operating at room temperature and pH close to neutrality2,5,6 MFCs could be one of the
most interesting technology for application in remote areas.
Despite for their terrific potential, only relatively few works have tried to demonstrate that MFCs can be used
as real power sources, as the pioneering research on MFCs integration on robotic platforms as the solely power
source7, and the demonstration of MFCs to power electronic systems8. Indeed, in the most part of current applica-
tions, focus is more given to strategies that can exploit other useful properties of microbial fuel cells, such as waste
water treatment or the production of valuable chemicals9, combining them to electricity generation in order to
increase the overall efficiency of the system. These approaches are advantageous if big plants are designed, while
strategies are currently needed to boost research on small scale MFCs, more emphasizing their key potential of
being easily movable from one place to another (i.e. portable) and potentially able to operate for long time, neither
with the need to be connected to the power grid, nor to the need of human intervention for replenishment (i.e.
autonomous)9.
In MFC technology, autonomy can be directly related to the density of the organic matter present into the
substrate used to feed the cells, and on its availability over time: the higher the latter parameters, the better the
performance and the lifetime of the MFC. Firstly introduced in 200110,11 using marine sediment as the source of
both bacteria and the organic matter, sediment microbial fuel cells (SMFC) demonstrated, for the first time, the
possibility to use solid phase organic matter at the anode and they are, up to now, the only class of MFCs able
to work autonomously for very long time12. SMFC can be designed both as large scale ones, to be used in sea
environment, or as small scale, portable systems13. Starting from that first example, the concept of SMFC has
been extended to the use of other solid phase anolytes as soil in plant MFCs14 and urban solid-wastes12,15, being
more generally referred to as solid phase MFC11,15,16 (SPMFC) even if the studies disregarding many often from
the concept of portable systems, considering that they often require feeding pumps17. In this communication we
propose a new approach to the design of small size microbial fuel cells in which the liquid anolyte containing car-
bonaceous and nitrogen sources is substituted by a new solid one, named synthetic solid anolyte (SSA), obtained
by means of the addition of agar. The key idea is to convert the liquid anolyte into a solid one, able to trap the
nutrients, granting an increase of their density and a slow release over time as it happens in natural solid phase
anolytes. We used the bacteria compatible Agar as the gelation agent, actually converting into a gel the liquid,

Center for Sustainable Futures, Istituto Italiano di Tecnologia, [Link] Trento 21, 10129 Torino, Italy. Correspondence
and requests for materials should be addressed to T.T. (email: [Link]@[Link]) or G.P.S. (email: gianpaolo.
salvador@[Link])

Scientific Reports | 6:29091 | DOI: 10.1038/srep29091 1

Figure 1. (a) The two chambers MFC used in this work: the inset shows the distribution profile of SSA
inside the anode chamber: the anodic electrode was lay down on SSA and immersed into buffer solution and
sandwiched between SSA and CEM membrane; (b) a picture of the agar based solid phase anolyte before use:
respect to traditional anolyte it embedded higher energy density and acted as a true energy storing system for
bacteria metabolisms and (c) the molecular structure of agar, gelling agent that permits to obtain SAA.

seawater based, anodic substrate. We placed the solid anolyte into two chambers reactors, and monitored the
performance of the resulting MFCs in terms of the open circuit voltage, short circuit current and power output.
While traditional liquid electrolytes need to be frequently replaced, the SSA-MFC showed a lifetime of 4 months
without the need of human assistance.

Results
Solid phase anolyte. A solid phase anolyte has been prepared mixing reagents that are common nutrients
for bacteria, such as glucose, fructose, peptone and sodium acetate, with a gelling agent such as agar (Fig. 1c).
Agar has been chosen because it is a natural polysaccharide, extracted from a group of red-purple marine algae,
well-known in biology as a culture medium of bacteria18, glucose and fructose, beyond to be fundamental nutri-
ent for bacteria, can act as cross-linking molecules during agar gelification giving a more compact solid and a less
shrinkage in case of dehydration19.
The gel has been prepared using a phosphate buffer solution, for preventing an acidification of the anodic
chamber that would be detrimental for the bacteria life.
A conductivity measurement of SSA has been performed in order to test the ion mobility inside the gel matrix.
The obtained value, 31.0 mS/cm, is not far from the result for the liquid electrolyte (27.6 mS/cm) having the same
composition save for agar.
Once the SSA was put into the cell, it was hydrated by means of the phosphate buffer solution. No further
liquids, with or without nutrients, has been added during the experiments.

Device performance. The experiments have been conducted in Microbial Fuel Cells consisting in two
squared chambers: the anode and the cathode, separated by a cation exchange membrane, as shown in Fig. 1a.
In the anodic chamber, the solid phase anolyte (Fig. 1b) fills the chamber for its whole volume, save for the
space necessary to place the electrode immersed into phosphate buffer solution. The cathodic chamber has been
filled by a liquid electrolyte (a phosphate buffer solution of potassium ferricyanide).

Scientific Reports | 6:29091 | DOI: 10.1038/srep29091 2

Figure 2. (a) Mean value of open circuit voltage (Voc) and current density (Jsc) recorded during the
electrochemical characterizations over time; (b) maximum power densities derived from the LSV curves;
(c) example of a LSV curve, collected in the 1st phase (day 7) and (d) power densities and voltages measured
under the application of external loads during the 2nd phase of the experiment (from day 15 to 30).

Among the experiments that were carried out, the results of the most complete in terms of characterization
and longer time span are presented. From the electrical point of view, the experiment has been divided into two
phases. The first phase was aimed at understanding the cell behavior under a constant low stressing load condi-
tion, while permitting the establishment of a stable electroactive biofilm at the anode20. In the second phase, from
day 15 to day 30, fifteen loads, from 330 to 8800 Ohm, from the highest to the lowest were sequentially applied, in
order to evaluate the performance of the cell covering different classes of applications.
Moreover, during both phases, the applied resistance was removed in order to perform electrochemical char-
acterizations such as Linear Sweep Voltammetry (LSV) and open circuit voltage measurements, after stabilization
period of open circuit voltage (OCV).
Results are shown in Fig. 2. As regards the cell performance in the first phase of the experiment, under the
1000 Ω load, the recorded cell voltage was about 0.5 V, with a contribution of the anodic potential of about −0​ .3 V
vs Ag/AgCl (−​0.1 V vs SHE).
This behavior demonstrates that the presence of the solid state electrolyte did not inhibit the establishment of
the proper reductive conditions at the biofilm/anodic material interface, with electrons being properly released
to the anode. The left side of Fig. 2a, obtained by polarization techniques, clearly shows that after a stabilization
period the VOC and Jsc values steadily increase, reaching the best values of about 0.8 V and 0.5 A/m2 respectively.
In the second phase, reported in the right side of Fig. 2a, the values of both VOC and Jsc slightly decrease, but are
quite stable. The power density trend has the same behavior that was noticed for VOC and Jsc, as shown by Fig. 2b.
The maximum power density value is closed to 60 mW/m2 recorded during day 7 (Fig. 2b,c). Interestingly, in the
second phase, even if the biological system was exposed to more stress because of the several loads applied in
a relatively short time, the output power density was quite stable and close to 45 mW/m2 (Fig. 2c). The average
power density and potential difference referring to each applied loads from day 15 to 30 are shown in Fig. 2d. At
both the highest and the lowest resistances (i.e., 8000 Ω and 330 Ω respectively), the minimum power density
values were obtained, that are 9.4 and 9.56 mW/m2 respectively. This behavior can be explained considering the
proximity of those values to OCV and SCC conditions, with potential difference values of 690 mV and 140 mV
respectively. For all values between 820 and 2150 Ω the power densities reach the highest values, with the maxi-
mum point of 35 mW/m2 at 1500 Ω, with a voltage of 580 mV and a current density of 61 mA/m2.

Energy efficiency of the SSA-MFC. Once the 30 days experiment was over, the MFC was opened and
the electrolyte was extracted. A provision of the possible MFC lifetime has been calculated by comparison of the
initial energy content in SSA and the energy produced, by MFC, during the experiment. The energy obtained after
one-month of operation running, named EMFC-OUTPUT, was calculated starting from the electricity generated by
applying loads during the time-course of the experiment, as described in Methods.
Instead, an estimation of the energy embedded into SSA before (Einitial) and after one-month operation (Efinal)
was made by calorimetric data, in terms of lower heating value. Therefore, the energy consumption (Econsumed) by

Scientific Reports | 6:29091 | DOI: 10.1038/srep29091 3

Figure 3. Energy evaluation of SSA-MFC: from the initial energy embedded into SSA (2013 J), to the
energy consumed (503 J), till the energy recovery by applying resistances over 30 days period (EMFC−
OUTPUT). The right axis shows energy density values in Wh/m . The values of energy are also shown in
2

percentage, in comparison to the initial energy present in SSA (100%). Energy efficiency, that is the ratio of the
useful energy recovered by MFC device to the heat of combustion of the SSA consumed, is about 63%.

means of microorganism’s metabolism could be deduced by the difference between Einitial and Efinal. The energy
recovery from 1-month experiment under loads is called EMFC-OUTPUT.
In order to estimate the energy really available for users, the total energy spent to run the device should be
taken into account. In this system, only cathodic replacement has an energy expenditure, considering that the
SSA-MFC device does not require in this configuration anodic feeding, mixing and temperature heating. The next
challenge will be to move toward a complete autonomous system thanks to energy and environmental sustainable
cathode, such as the air-cathode that will be coupled with the SSA21,22. Figure 3 depicts the energies involved in
SSA and their ratio respect to the energy embedded in SSA, considered hence as 100%.
As shown in Fig. 3, only a quarter of energy was consumed during the 30 days experiment (Econsumed =​ 503 J)
and therefore, it was possible to estimate a total working time of the SSA-MFC of at least 4 months in continuous
working condition as a self-sustainable energy harvester.
Taking into account the EMFC-OUTPUT from 1-month experiment under loads (~251 J) and the chemical
energy consumed by bacteria in that period (~503 J), it was possible to evaluate the energy efficiency (EE) of the
SSA-MFC that reaches 63% (details of the used formula in Methods).

Discussion
The gel prepared as described in the method paragraph, can be considered a solid state electrolyte, even if inside
its framework a liquid phase is still present, because it is well accepted by the scientific community considering
gels as solid electrolytes23,24.
In order to estimate the resistances of ions conductions inside the solid electrolyte, the ohmic resistance has
been evaluated. The estimation of the Ohmic losses (RΩ) arising, among other factors, from the resistance of ions
conduction into the electrolyte and the membrane, and of electrons conduction through the electrodes25. It was
carried out by means of the current interrupted method (CIM), as described in Methods (MFC operation and
characterization). The average value of (12 ±​ 2) Ω was calculated considering resistances from both the first and
the second phase. This result is quite interesting and helps in clarifying that the presence of the new introduced
solid electrolyte does not increase the RΩ value. The obtained Ohmic losses are, comparable with those typically
measured for liquid anolyte with the same boundaries conditions26,27. Once the experiment was over it was possi-
ble to notice that the SSA was less compactness than it was at the beginning of the experiment. This phenomenon
can be explained by the contribution of two different processes: one can be related to the glucose and fructose
consumption due to the bacteria, that causes the reduction of the cross-linking degree among the agar molecular
chains, while the other processes can be related to an agar degradation due to the seawater bacteria inoculum,
because many marine microorganisms can degrade this algal compound by production of agarase enzyme. In
particular the presence of craters on the SSA surface, observed at the end of experiment is a result of the enzy-
matic digestion of the agar18.
The EE, defined as the ratio of energy recovered by the MFC to the heat of combustion of the consumed
organic substrate28, gives important information on the energetic performance of the device. Moreover, since
it takes into account energy losses due to external devices, such as the pumps for feeding and recirculation28,
it can help appreciating the unique features of the SSA-MFC. Indeed, not needing hydraulic pumping systems
for anodic feeding, the SSA-MFC has a zero energy contribution from external systems at the anode, helping
to obtain an overall positive energy balance29, with an impressive value of EE close to 63%. It is important to
say that since this study was mainly focused to analyze the behavior of the new SSA, and hence at this stage the
repletion of catholyte, in terms of energy costs, has not been taken into account, because the focus is on the

Scientific Reports | 6:29091 | DOI: 10.1038/srep29091 4

anodic chamber energy balance and not that of the whole MFC. The choice of a two-chamber MFC with a liquid
chemical catholyte, instead of the open-air cathode architecture is to focus the attention mainly into the new SSA.
Considering the difficulty to estimate heat of combustion of wastewater, in literature the results usually show the
Coulombic Efficiency (CE) that represents the conversion of organics into electrical charge.
However, CE does not take into account the energy expenditure to run the device and hence, it is not able to
give indication of how far MFCs are from an energy-positive device29. Data present in literature, show CE values
ranging from 1% to up 80%28–33. These variations mainly depend on the organic substrate but also on design,
operative conditions and microbiology.
It is evident, therefore, that, in many cases, if energy consumption of the external devices were taken into
account, these efficiency values would be dramatically lower.
The present work fixes a new important step toward the development of a solid phase anolyte and its capability
to function as nutrient storage available for bacteria and convertible into useful energy in the time, without any
external nutrients addition. It acts as true chemical energy storing system able to release slowly its energy over
time and significantly contributes to the autonomy of the resulting system. The easy preparation of SSA and the
easy uptake of it from bacteria reveal that solid SSA in an original and convenient fuel for MFC. This is an impor-
tant step toward the reduction of energy request, bringing a better net energy balance necessary to overcome the
biggest bottleneck to marketability of MFCs. The convenience of maintaining a growing community of bacteria
thanks to a high-density energy substrate could mean a potentially long-term source of energy with numerous
applications: from remote energy source to the waste management practices as treatment of both water and solid
waste. Taking into account that many electronic devices or sensors, i.e. temperature sensors, require 1.5 V as oper-
ative voltage or even less, i.e. piezoresistive sensor26 require ~1 V, one possibilities is to design a system at least of
two MFCs connected in series and eventually with a capacitive storage system. The remote application could take
advantage from natural environments, i.e. seawater. There bacteria can found a richer substrate releasing more
electrons, permitting to power sensors in remote area. Moreover, the high efficiency of the device (63%) together
with the zero contribution for anodic feeding is a crucial step toward a new clear route to portable and autono-
mous MFC devices for remote applications, cutting down, significantly, operative costs, energy requirements for
the system maintenance and reducing hydraulic problems.
It is worthwhile to underline that the anodic chamber, after it was sealed, did not need any more feeding or
hydration during the experiment neither reinoculation, giving rise to a really autonomous device from the anodic
side.
Next important step could be the integration of air-cathode to replace the chemical catholyte, in order to push
the research toward a complete autonomous system. The non-astonishing performance have not to restrain the
opportunity of a further development, because SSA shows the way for a new generation of MFCs, that can, for
example, be, realistically, employed in devices for remote areas or harsh environments monitoring, powering
small devices, such as sensors.

Methods
MFC configuration. The MFC devices consist of two squared chambers: the anode and the cathode. Both
compartments were made in Poly(methyl methacrylate) (PMMA) with dimensions 8 cm ×​ 8 cm ×​ 2 cm and sep-
arated by a cation exchange membrane (CEM, CMI 7000, Membranes International Inc.), as shown in Fig. 1a. A
square piece of graphite felt (64 cm2) (Soft felt SIGRATHERM GFA5, SGL Carbon, Germany) was introduced in
both anode and cathode chambers and connected with a graphite rod (5 mm in diameter) to ensure an effective
current recovery. The solid phase anolyte contains in: 8 g·L−1 sodium acetate, 7 g·L−1 glucose, 7 g·L−1 fructose,
10 g·L−1 peptone, 25 g·L−1 agar, 8.2 g·L−1 Na2HPO4·2H2O and 5.2 g·L−1 NaH2PO4·H2O dissolved into diluted sea-
water (ratio seawater: distilled water =​ 1:4). The solution, after being autoclaved at 125 °C for 15 min was poured
into plastic mould, becoming solid overnight. The conductivity of SSA of 31.0 mS/cm was measured by WTW
Multi 3430 multimeter (Germany).The electrolyte is, afterwards, released from the mould and placed into the
anodic chamber. The gelling agent used, agar (Fig. 1b) is a polysaccharide, obtained from particular types of
seawood and among the advantages that if offers for use in microbiology, the principle one is that it remains solid
at normal incubator temperature, from 4 to 45 °C. In Fig. 1c is shown the picture of obtained SSA. Then, the SSA
was inserted inside the anode chamber covering whole its volume saved for the carbon felt that was laid down
upon it and the graphite rod and immersed into buffer solution. MFC was inoculated in the anode chamber by
sea water (Arma di Taggia (IM), Italy), previously enriched with following cultures (in three steps) in anaerobic
conditions. The experiment was performed at room temperature (24 ±​ 2 °C). The mixed culture, before being
inoculated in MFCs was previously enriched in batch and under gentle orbital shaking (150 rpm), with the same
medium used for SSA except for the absence of agar. This medium used for bacteria growth and inside the MFC
is less selective in order to allow growing of resident marine consortia. Seven ml of enriched sea-water inoculum
was directly distributed on carbon felt surface. To prepare anodic and cathodic solutions, a phosphate buffer, i.e.
Na2HPO4·2H2O (8.2 g·L−1) and NaH2PO4·H2O (5.2 g·L−1) was used. The cathodic compartment was filled by
potassium ferricyanide (6.58 g·L−1) used to perform reduction reaction. The electrolyte was replaced two times a
™
week by syringe pump (NE-300 Just Infusion ​Syringe Pump, USA) during the experiments to ensure a constant
behavior of the cathodic side of the cell. Regarding the anode, no need of replenishment or reinocultaion during
all period of test was necessary, at all.

MFC operation and characterization. From the electrical point of view, the experiment was divided
into two phases (Fig. 2): during the first one (lasted from day 0 to day 15), the MFCs were investigated under
a constant value of electrical loading (1000 Ohm). In the second phase (from day 15 to day 30) fifteen loads,
from 330 to 8800 Ohm, from the highest to the lowest were sequentially applied (one per day. The cell voltage
(Δ​V) under external resistors was continuously measured every 60 s using a data logger (Agilent, 34972A, The

Scientific Reports | 6:29091 | DOI: 10.1038/srep29091 5

Netherlands). During both phases, two-times a week, the external applied resistance was removed in order to per-
form an electrochemical characterization analysis, by means of a multi-channel VSP potentiostat (Biologic), by
varying 1 mV/s potential from open circuit voltage to short circuit current conditions (linear sweep voltammetry
technique-LSV), after a stabilization period (few minutes) under OCV conditions. Measurements were recorded
®
by using EC-Lab ​software version 10.1x (BioLogic) for data acquisition. Before starting the electrochemical
measurements, the cathodic electrolyte was replaced by a fresh one. The cell performance was investigated cal-
culating the power produced by the device for each load, by applying the Joule’s Law, P =​  V2/R, where R is the
external resistance and V is the correspondent collected voltage between the anode and the cathode. The current
interrupt method (CIM) was performed to determine the internal resistance of the MFC through the interruption
of the current flow at fixed potential of 0.3 V and the observation of the resulting voltage transients. The test was
carried out using a perturbation to the system with a very short duration (50 ms) in order to obtain precise and
accurate determinations.
A calculation of the energy before (Einitial) and after one-month operation (Efinal) was made considering the
heat of combustion of the organic substrates embedded into SSA (in particular the lower heating value was taken
into account). This measure was performed by isoperibolic calorimeter (Parr 6400, FKV Italy). By the difference
between Einitial and Efinal the energy consumed by bacteria’s metabolism in one month (Econsumed) was obtained.
In order to estimate the energy produce by MFC during the experiment, Δ​V acquired by data logger was use to
calculate the current (I) from Ohms law: I =​  V·R−1. Thus, energy produced (EMFC−OUTPUT) by SSA-MFC in Joule
(J) during one-running month was calculated as the integration of the electrical power in the time (t) by the
following equation:
t final
E= ∫t initial
I ⋅ ∆V ⋅ dt
(1)
From these data, it was possible to estimate the energy efficiency (η​) as the ratio between the E MFC−OUTPUT and
the energy consumed by microorganisms from SSA in one month:
E MFC − OUTPUT
η (%) = ∗ 100
ECONSUMED (2)

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Acknowledgements
The authors acknowledge Prof. Bernardo Ruggeri and Prof. Elena Tresso of Politecnico di Torino for the valuable
scientific discussions. Furthermore, we thank Dr. Adriano Sacco and Dr. Valentina Margaria from Istituto Italiano
di Tecnologia, Dr. Carla Celozzi and Dr. Vanni Martignoni from Politecnico di Torino for their contributions on
data analysis. The research was supported by internal funds.

Author Contributions
T.T., G.P.S. and M.Q. conceived the idea. T.T. designed experimental set-up and methods. T.T. and G.P.S.
developed the solid phase anolyte, performed experimental tests and analysed the data. All the authors
contributed to discussion and manuscript preparation.

Additional Information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Tommasi, T. et al. New insights in Microbial Fuel Cells: novel solid phase anolyte.
Sci. Rep. 6, 29091; doi: 10.1038/srep29091 (2016).
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Scientific Reports | 6:29091 | DOI: 10.1038/srep29091 7