MFC-Cascade Stacks maximise COD reduction and avoid voltage reversal
under adverse conditions
Pablo Ledezma1, John Greenman2 and Ioannis Ieropoulos1*
1
Bristol Robotics Laboratory, Universities of Bristol and of the West of England, Frenchay Campus,
Bristol BS34 8QZ, U.K. 2 Department of Applied Sciences, University of the West of England, Bristol
BS16 1QY, U.K.
* Corresponding author: Ioannis.Ieropoulos@brl.ac.uk
Telephone: +44 (0) 117 32 86318
Fax: +44 (0) 117 32 83960
Abstract
This study presents a 6-unit continuous flow Microbial Fuel Cell (MFC) stack organised as a
vertical cascade and tested under different electrical configurations. Under parallel
conditions, the stacked-system demonstrated stable operation despite the substrate
imbalances inherent to cascading and higher power and current densities than individual
MFCs (positive stacking effect), whilst the cascading dynamic allowed for a cumulative COD
reduction of >95% in less than 5.7h, equivalent to a very competitive 7.97 kgCOD m-3 d-1.
When changed to a series configuration, the stack exhibited considerable losses (crossconductance) unless correct fluidic/electrical insulation of the units was applied; with this
problem resolved, the stack in series also exhibited electrical stability and performance
superior to individual cells. In both electrical configurations, the 6MFC system was
purposefully deprived of feed repeatedly for up to 15 days, and in no case resulting in
significant losses post-starvation. The combined results of 14 months of experimentation
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
hereby presented, demonstrate that cascade-stacking of small units can result in enhanced
electricity production (vs single large units) and treatment rates without the need for
expensive catalysts. Moreover, it is also demonstrated that MFC operation (even when the
continuous flow is stopped) in series configuration, substrate imbalance and starvation does
not necessarily result in cell-voltage reversal.
Keywords
Microbial fuel cells; stacking; wastewater treatment; voltage reversal
Introduction
Microbial Fuel Cells (MFCs) have been receiving increased interest in the last three decades,
since the production of electricity, from a wide variety of organic wastes, was demonstrated
without the need for artificial mediators (Habermann & Pommer, 1991). MFCs are thus an
attractive alternative for effluent treatment (Clauwaert et al., 2008) and despite the
technological progress, it is clear that current bench top MFCs (particularly individual
systems), cannot cope with the volumes and flow rates needed to viably operate in a
wastewater treatment plant. Scaled-up systems are therefore needed to allow for efficient
and high treatment of such large volumes, but also to be able to produce sufficient
electricity so that their operation for wastewater treatment can be made autonomous
(Clauwaert et al., 2008; Winfield et al., 2011).
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
However, scaling-up via increasing reactor sizes carries the inherent challenge of increased
internal resistances (Rint), since this is volume-dependent (Dekker et al., 2009; Ieropoulos et
al., 2008). This is probably one of many factors that could explain why large volume MFCs
have not demonstrated high-power output levels as smaller bench systems have. In fact,
power density has been demonstrated to be higher at the smaller scale (Ieropoulos et al.,
2008), meaning that practical (useful) levels of power from MFCs are more likely to be
generated via miniaturisation and multiplication, rather than enlarging single reactors
(Fornero et al., 2010; Ieropoulos et al., 2008; Ieropoulos et al., 2010a; Kim et al., 2011). In
terms of MFC unit stacking of various sizes, there have been numerous reports in the
literature, (Aelterman et al., 2006; Dekker et al., 2009; Ieropoulos et al., 2003; Ieropoulos et
al., 2005; Ieropoulos et al., 2010a; Ieropoulos et al., 2010b; Melhuish et al., 2006; Oh &
Logan, 2007; Shimoyama et al., 2008; Shin et al., 2006; Wang & Han, 2009; Wilkinson, 2000;
Zhuang & Zhou, 2009; Zhuang et al., 2012), however much less attention has been paid on
the cascade element of multiple MFC units in a stack operating under continuous flow
conditions (Aelterman 2006; Ieropoulos 2008, Galvez 2009, Pinto 2010, Winfield 2011) and
how the losses from direct fluidic paths oppose electron flow (Ieropoulos 2008, Zhuang &
Zou 2009; Shuang et al 2012). The aim of this paper is therefore to address the issues of
fluidic conductance in a continuous flow cascade system and demonstrate improved
operation both in terms of power output as well as treatment efficiency.
Complexity is further increased when considering the peripherals required for the operation
of MFC stacks (Ieropoulos et al., 2009). According to a life-cycle evaluation of MFCs, which
has found that fabrication costs should be brought to a minimum (i.e. no expensive
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
catalysts) (Pant et al., 2011), it is clear that the inclusion of additional electronic components
in the operation e.g. DC-DC converters (with their inherent losses) can only increase system
costs and thus reduce the competitiveness of the technology. Accordingly, the most
inexpensive way to achieve working voltages and currents is through a combination of series
and parallel configurations. Yet this is not without problems, with cell-voltage reversal being
one of the most commonly reported phenomena for stacks in a series configuration.
Probably observed for the first time in MFCs by Aelterman et al. (2006), this incident was
then further investigated by Oh & Logan (2007), with unbalanced substrate concentrations
and starvation reportedly being the main causes of reversal. Further investigation of MFC
stacks in series has since been largely neglected with a few exceptions (Dekker et al., 2009;
Ieropoulos et al., 2008; Kim et al., 2012; Pinto et al., 2010). During this time the consensus
has been that series connection, under continuous flow, almost always implies cell voltage
reversal (Kim et al., 2011; Lefebvre et al., 2011), despite the fact that in the initial studies,
this was not the case (Ieropoulos et al., 2008; Oh & Logan, 2007). Since wastewater
treatment systems operate in continuous or semi-continuous mode, perhaps there should
be no hesitation in using, at least partially, series connections within the stacks to increase
voltage to usable levels. Moreover, Pinto et al. (2010) have demonstrated that MFCs with
anolyte staging connected fluidically in series, are capable of larger treatment capacity than
the same cells in (fluidic) parallel configuration. Enhanced treatment is of particular
importance, especially since MFCs might find their first commercial application in
wastewater treatment, as currently the market value of the electricity produced at low
performance levels is considerably lower than the added value of the environmental service
of treating the waste (Fornero et al., 2010).
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
The present investigation follows on from all these observations and previously
demonstrated stable stack operation (Ieropoulos et al., 2010b) and proposes a simplified,
inexpensive solution to stacking for maximising treatment without parasitic crossconduction or voltage reversal losses.
Materials and Methods
1. Construction of the MFC-Cascade system
A cascade-type MFC system was modelled using Solid Edge v20.0 UGS PLM Software
(Siemens, Germany). The design incorporated both an upflow siphon-type tubing system
(embedded) that allowed continuous flow whist maintaining a constant internal electrolyte
volume, in addition to a dripping mechanism that created a fluidic isolation and thus
electrical insulation between MFC units of the cascade. In such a system, the fluid flow was
mostly gravity driven, with the need to pump only to the fuel cell at the top.
The MFC units were also designed to allow for continuous flow of nutrients even in the
event of channel blocking or the absence of cells in the cascade (for e.g. maintenance). The
design was then analysed for structural reliability and mouldability with the ProtoQuote®
analysis system (ProtoMold Injection Moulding Service, Shropshire, UK) and modified to
guarantee that the design could be fabricated by Rapid prototyping techniques as well as
Injection Moulding, since the latter can dramatically reduce the costs of fabrication. Once
finalised, the design was used to make, by means of Fused Deposition Modelling (FDM
Titan, Laserlines, Bedford, UK), packs of 3 identical 2-chamber MFCs (each triplet in a
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
containing unit, see Fig. 1) with 6mL internal volume for each half-cell in ABS (Acrylonitrile
Butadiene Styrene) plastic. The electrodes, both anode and cathode, were made of 180 cm 2
of 20 g.m-2 plain carbon fibre veil (PRF Composites, Dorset, UK) without any added precious
metals or catalysts; the current collector for each electrode was made of plain NiCr wire (6
cm long, ø0.45 mm; SWC, Essex, UK). A cation-exchange-membrane with a surface area of 6
cm2 (VWR, Leicestershire, UK) was placed between the two chambers. The original inoculum
was activated sewage sludge provided by the local water utility company (Wessex Water
Services Ltd). Two single MFCs with identical dimensions were set up at the same time and
operated independently as controls.
Taking advantage of the precision in the fabrication technique, along with the predictable
hygroscopic swelling of the parts (avg. +0.399% according to the manufacturer), the MFCs
were watertight without the need for screws, fixtures or sealants thus reducing assembly
time, complexity, cost and weight of the stacks. Consequently, the utilised design conveyed
highly practical “plug-and-play” modularity, whereby each MFC could be easily added to a
stack or removed for e.g. maintenance, both in a matter of seconds and without affecting
the downstream flow for the rest of the stack.
2. Operation
The stacks and control MFCs were continuously supplied with sterile 5mM Acetate TYE
anolyte (5mM C2H3NaO2, 0.1% w/v Tryptone, 0.05% w/v Yeast Extract; pH 6.8 unbuffered,
680 ± 20 μS.cm-1) at 120 µL.min-1 by a multi-channel peristaltic pump (Watson-Marlow
Pumps Group, Cornwall, UK), equivalent to an organic loading rate (OLR) of 8.366
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
kgCOD.m3.day-1 and a hydraulic retention time (HRT) of 0.63h (per MFC in the cascade). In
additional experiments, the anolyte was replaced by an artificial wastewater solution as
formulated by Aldrovandi et al. (2009), without buffering. The catholyte was fresh tap water
in all cases.
The external load (Rext) connected to each MFC or Stack was varied over time in an attempt
to achieve impedance-matching (Rext = Rint ), which according to Jacobi’s law, is the condition
under which maximum power is transferred to the external load, a.k.a the Maximum Power
Transfer (MPT) point. By repeatedly testing the MFCs/Stacks using polarisation — standard
technique with a range of external resistors (30kΩ to 50Ω) and 5min steps with a variable
resistor box (ELC, Annecy, France) — it was possible to observe which Rext values produced
MPT. These same values (which decreased over time, indicating a reduction of R int as
previously described by Ieropoulos et al. 2010b) were then utilised as the load in the normal
operation of the cells, leading to continuous MPT over time.
Additional experiments involved the connection of the MFCs in series and parallel
configurations; the resulting stacks were tested for stability by different cycles of starvation
brought about by stopping and re-starting the anolyte pump for different periods of time.
All tests were carried out at ambient temperature in an attempt to simulate a pragmatic
natural environment for a period of 14 months.
Polarisation and power curves for stacks and single units were generated under the same
continuous flow by connecting the MFCs to load values ranging from 50000 to 5Ω (at 3min
intervals, found sufficient to allow the MFCs to reach quasi-steady-state for each resistance
value).
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
3. Data collection and analysis
3. 1
Electrical output
Electrical output was measured in real time in the form of voltage with a PicoLog ADC-16
interface (Pico Technology, Cambridgeshire, UK) connected to a computer. Current
production was calculated using Ohm’s law by which the current I = V/R , where V is the
measured voltage and R is the load. Electrical power production was determined using the
derivation of Joule’s law where the power P = V x I. Power and current densities were
normalised based on total anodic compartment volume (6cm3, multiplied by the number of
MFCs when stacked).
3. 2
COD measurements
The total COD of samples was determined according to the potassium dichromate oxidation
method with a kit from VWR (COD MR test system, Lutterworth, UK) and using a compatible
photometer (Aquagem, Jenway, Chelmsford, UK) and interference filter module (COD MR
IM, Jenway, Chelmsford, UK). Samples were collected at the outlet port of each MFC and
compared with the supplied sterile input anolyte (Acetate TYE or Artificial wastewater).
Results and Discussion
1. Individual operation
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
Originally, a single triplet (3MFCs; see yellow triplet in Fig.1) and two single reference MFCs
were inoculated and operated until a steady-state (based on voltage output) was observed
for a period of at least 7 days; this took approximately 3 weeks in the case of the triplet. It
was soon realised that 3MFCs were not sufficient for full nutrient utilisation/treatment (data
not shown). Hence, a second triplet was placed immediately below thus making a cascade of
6 MFCs, but still fed only from the drip at the top of the cascade. However, the new triplet
was not inoculated with activated sludge but just left to be colonised by the cells shedding
off from the MFCs placed above them.
In less than 24h, a clear electrical potential rose in these newly added MFCs (data not
shown). The system was tested continuously for a further 3 weeks until steady states were
reached for all 6 MFCs. The power curves obtained at the end of this period (see Fig. 2)
indicate a pattern, whereby the fuel cell placed at the top of the cascade (MFC1) produced
the maximum power and current densities, and the fuel cells downstream produced lower
values, in a linear relationship with their position in the cascade; a significant correlation
was actually found between the maximum power density (MPD) points for each MFC (see
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
insert table on
4
Power density [W.m
-3
]
-3
-3
Cell MPD [W.m ] CCD [A.m ]
MFC1
3.69
14.32
MFC1
MFC2
3.23
13.39
MFC2
MFC3
3.12
13.16
MFC3
MFC4
2.87
10.43
MFC4
MFC5
2.47
7.67
MFC5
MFC6
2.19
7.22
MFC6
3
2
1
0
0
10
20
30
Current density [A.m
40
-3
50
]
Fig. 2) and the corresponding current (CCD) with regards to the actual positioning (linear
regression: r2= 0.9296; p= 0.0019). Similarly, the cells presented differential open circuit
voltages (OCVs) in linear proportionality (r2= 0.9472; p= 0.0011), with MFC1’s OCV = 584.76
mV and 576.70, 567.22, 528.47, 511.97 and 503.32 mV respectively for MFCs 2-6.
Nevertheless, MPT for all cells was found to occur at very similar values (avg. 3.27 ± 0.21 kΩ)
for all cells, indicating near-identical Rint. The latter could be expected because all MFCs
were of identical in construction, so the differences in OCV can be explained by the
cumulative degradation of the nutrient supply, brought about by feeding the stack only at
the top cell; as the anolyte flows from one cell to the other, electrons are extracted by the
bacteria, thus changing the anolyte’s redox potential with direct consequences for the
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
anodic potential of the cells. Furthermore, but to a lesser extent, it is also possible that a
cumulative reduction in the catholyte’s dissolved oxygen also contributed to this effect.
2. Stacked operation
2.1 Parallel connections: positive stacking-effect
After estimating the individual performance of each cell in the stack, the cells were
connected in parallel and left to stabilise; as expected the OCVs for both the stack and the
individual MFCs were very similar (ca. 560mV). After a week of stacked operation, a
polarisation sweep was performed, with the results shown on Table 1a. As can be seen, the
6MFC stack was capable of producing approx. 7 times more power than individual MFCs,
implying a superior power density from the pack. In a similar fashion, the MCP was 8.33-fold
larger than the single units’ production. After another ~8 months of operation in parallel
(see Table 1b), both individual and stacked systems exhibited improvement, but the
superior performance of the stack was maintained with respect to the reference cells (7.46
W m-3 and 91.11 A m-3 vs 6.83 W m-3 and 85 A m-3), implying that this effect is permanently
maintained throughout biofilm development stages. The benefits of stacking – when this is
done optimally – are thought to be a consequence of impedance matching (see above),
whereby the plurality of units tends to uniformity in terms of R int. If the majority of the cells
perform well then the negative effects of the underperforming minority are compensated
for, and in the longer term their performance is “assisted” by, the stronger majority. If the
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
opposite case occurs, then the overall performance is brought down (Ieropoulos et al.,
2010c). The importance of Rint balancing is further explored in section 2.5.
2.2 Parallel connections: cumulative COD reduction to high standards
2.2.1 COD reduction for Acetate-based anolyte
The COD-reduction was measured both for the individual reference cells and for each cell of
the stack operating in parallel, with the latter being the preferred configuration due to
higher currents obtained. The ΔCOD levels achieved were based on the COD of the sterile
Acetate TYE anolyte (1982.5 ± 2.12 mg L-1). Determinations were performed until the CODreduction values became stable (approx. 3 months after connection in parallel; see Fig. 3
“5mM Acetate + TYE 1”). A second set of determinations were performed one month later
(see Fig. 3 “5mM Acetate + TYE 2”), but no significant difference was found. The average
ΔCOD for the top-placed MFC1 (cascade) was 591 mg L-1, whereas the ΔCOD for a nonstacked MFC was on average 542 mgCOD L-1. The difference observed can be explained by
the positive stacking-effect previously discussed: operation at higher current densities
means enhanced abstraction of electrons from the carbon source, naturally translating into
better COD reduction rates than in individual units.
The average ΔCOD for the 6MFC-cascade stack was found to be 1753.5 mg L-1 or 88.45%,
producing an effluent of 229 ± 8.48 mgCOD L-1. The latter, although favourable, was
nevertheless of insufficient quality, particularly with reference to the European Union urban
wastewater treatment directive (EU WWTD), which requires COD reduction to a minimum
of 125 mgCOD L-1 (EEC, 1991). As ΔCOD was found to be cumulative in the cascade system,
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
it became clear that adding further units below the stacked system would assist in reaching
acceptable levels. However, due to the impracticality of setting up a second 6-MFC stack —
with comparable levels of biofilm maturity — within a reasonable timeframe, the addition of
a second 6MFC stack immediately below was not immediately feasible. Recirculation of the
effluent was also not suitable due to the long HRTs employed (degradation of the substrate
by planktonic bacteria would have continued regardless, so the feedstock would have been
inconsistent, with continuously decreasing COD over time). Further degradation capabilities
were thus ‘simulated’ i.e. estimated by switching the feedstock, at the top of the cascade, to
a concentration of ~230 mgCOD L-1 (obtained by the dilution of 116mL of 5mM Acetate TYE
into 1L of distilled water). This may have not been the most ideal simulation scenario, since
it eliminated the bacterial component from the feed, however it was one practical way of
guaranteeing constant COD feeding for prolonged periods of time at the levels exhibited by
the original stack’s effluent. The combined results of COD degradation for the 6MFCs at ~2
gCOD.L-1 and then 6MFCs at ~230 mgCOD L-1 are presented in Fig. 3.
With the ‘simulated’ addition of 3 more MFCs, ΔCOD reached 95.3%, while no significant
difference (in percentage terms) was found when the anolyte passed through more cells
(see Fig. 3: MFCs 10, 11 and 12). With the presented operating conditions and OLR, a
cascade of 9 fuel cells would have thus been sufficient to obtain an effluent of acceptable
quality in terms of COD (effluent: 92.5 ± 6.36 mgCOD L-1) and a very competitive removal
rate of 7.97 kgCOD m-3 day-1 (Tchobanoglous et al., 2003).
2.2.2 COD reduction for Artificial wastewater
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
The artificial wastewater displayed a COD value of 164 mg L-1 for an OLR of 1.04 kgCOD m-3
d-1. Despite the lower concentrations, a wastewater of such levels would still be above EU
WWTD regulations therefore requiring treatment before release. When this artificial
wastewater was used as the anolyte feed, reduction was found to be 39.1% for the 6MFC
stack (ΔCOD 64± 3.61 mgCOD L-1), resulting in an effluent with ~100 mgCOD L-1 (see Fig. 3).
This is satisfactory in terms of COD, and in fact it was found that for such low-concentration
effluents, a 3MFC-staging was sufficient to reach adequate levels (ΔCOD: 31.7%, effluent:
112±5.0 mgCOD L-1).
2.3. Series connection: the need for electrical insulation
After the parallel configuration (see Fig. 4a) experiment was concluded, the stack’s electrical
connections were switched to a series configuration, with all the other experimental
conditions remaining the same. The OCV of the stack was found to be ~2,270 mV, which
was considerably less than expected (3275.44 mV based on multiplying by 6 individual
OCVs). This drop has also been observed in other investigations (Aelterman et al., 2006;
Ieropoulos et al., 2008; Wang & Han, 2009; Winfield et al., 2011; Zhuang & Zhou, 2009;
Zhuang et al., 2012) and linked to ionic cross-conduction between cells’ anodes (conductive
electrolytes create an electric bridge between anodes, but these are also linked via external
cables to cathodes due to series configuration), creating “antagonistic paths” that negatively
affect electrical performance. In the current study this effect was not caused by the anolyte
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
(fluidically/electrically insulated by air gaps) but by the catholyte, which was not flowing in
an isolated manner; this is illustrated in Fig. 4b. The tap water conductivity was ~680 μS cm1
compared to ~350 μS cm-1 for the anolyte, so the effects of cross-conductance could be
anticipated to be more deleterious than in the case of anolyte cross-conduction.
Experiments under electrical load showed that the power production was 97.6% lower than
that produced under parallel-connection mode (MPD: 0.18 W m-3; MCD: 0.55 A m-3). The
system was left to operate for one further month under these conditions, but no
improvement was observed (MPD: 0.13 W m-3; MCD: 0.55 A m-3), emphasising that this was
not a transient effect i.e. change of anodic potentials affecting biofilms.
To resolve this cross-conduction problem, other investigators (Kim et al., 2012; Winfield et
al., 2011; Zhuang & Zhou, 2009) have focussed on increasing the separation distance in
fluidically linked MFCs. In both cases, a larger separation seemed to reduce, although not
completely eliminate, the negative effects of anolyte cross-conductance. The separation
distance (originally 15 cm) in the present study was accordingly increased up to 250cm but
no significant improvement was observed (data not shown).
When a true air-gap system was introduced for the cathodes identical to the one for the
anolyte system (see Fig. 4c), power output increased more than 27-fold (MPD: 4.86 W m-3),
demonstrating the positive effect of fluidically/electrically insulating the MFCs in the
cascade. This improved performance was nevertheless inferior to the average production of
reference individual cells (MPD: 6.83 W m-3). Following two months of continuous
operation, the performance of the stack surpassed that of the reference cells,
demonstrating that fluidic isolation in a series connected cascade is fundamental for
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
improved operation. As shown in Fig. 5, the air-gap isolated Cascade-stack produced similar
levels of power output as when connected in parallel (in accordance with the laws of Joule
and Ohm), but in fact achieved an overall maximum power density of 9.29 W m-3 (vs 6.83 W
m-3 for individual cells) and a current production of 15.55 A m-3 (higher than the expected
MCD 85 A m-3/6MFCs = 14.17 A m-3) with an identical OCV (3253.12 mV vs 3275.44 mV) to
the mathematical sum of the individual OCVs presented in part 1.
These results clearly demonstrate that it is possible to operate stacks in series without the
voltage losses observed by others and with the added advantage of the positive stackingeffect (higher power and current densities than for single cells; see Fig. 5), provided that
correct electrical insulation is employed (Fig. 4c). The latter was critical in the present study
for optimum performance, which is in contrast to Dekker et al. (2009) who stated that no
electrical insulation is required for adequate stack performance.
2.4. Series connection: no reversal under substrate imbalance
The cascade-MFC system presented herewith operated under substrate imbalance
conditions by default, since each of the cells in the stream receive a completely different
concentration of nutrients, as shown in Fig. 3. Even under these adverse conditions of nonuniformity, in a period of >6 months of continuous operation in series, no voltage reversal
was observed (even when the stack was subject to the electrical shorting described in 2.3).
These results are in agreement with Zhuang et al. (2012) , whereby a 5-stage MFC stack
connected in series and with imbalanced substrate concentrations never showed reversal.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
Both investigations demonstrate that substrate imbalance is not the cause for reversal in
MFCs. The electrical reasons for cell reversal are discussed in the next section.
2.5. Series and parallel connections: resilience to starvation
The results of the starvation cycles introduced in-between the several months of testing are
depicted in Fig. 6. In parallel configuration, the stack showed the capability to fully recover
to pre-starvation current production levels in single-dip (see B, Fig. 6a), double-dip (see A,
Fig. 6a; both of ~100 h) and long-term deprivation cycles (~15 days for cycle C). In series
configuration (see cycles D-G, Fig. 6b), full recovery of power levels was also achieved after
every cycle of starvation, independent of the length of time. Nutrient depletion was shown
to have no adverse effects on the performance and in addition it did not affect the
continuous improvement of the stack’s output (2.60 to 7.25 W m-3) after the electrical
shorting described in 2.3. These results demonstrate that starvation does not necessarily
lead to long-term deleterious effects as previously reported (Oh & Logan, 2007). It has long
been known that nutrient-deprived bacteria switch reversibly to a state of metabolic arrest
(Amy & Morita, 1983). This is also the case for biofilms, but the latter tend to be more
resistant to starvation than planktonic cultures, amongst other adverse conditions (LappinScott & Costerton, 2003). Starved biofilms have been shown to express starvation-specific
genes, which can bring cells to a state where little or no growth occurs (Chávez de Paz et al.,
2008). Since near-identical levels of production were found post-starvation for all cycles
(See Fig. 6), and recovery was observed in a period of time equivalent to <3 volumetric
replacements (~8 h in all cases except for C; 3.79h for a full volumetric replacement at set
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
flow rate), it is hypothesised that the grand majority of the cells in the biofilms remained
viable during the deprivation cycles but were dormant, as recently shown in simulations by
Ayati & Klapper (2012). Further research is needed to verify whether this is the case and
also to understand why the recovery in cycle C took considerably longer (ca. 47 h, perhaps
involving recolonisation/replacement of dead cells) but still resulted in levelled performance
(12.52 A m-3 pre vs 12.40 A m-3 post-starvation).
It should be noted that the Cascade-MFC design allows for synchronous effects in
starvation: when the feed is stopped at the top, all of the cells are starved at the same time,
which allows for a “balanced” drop in bacterial activity that is thought to help maintain all
cells’ potentials positive. Localised (“imbalanced”) starvation has not been deeply studied in
MFCs, but on PEMFC stacks it has been shown to cause reversal and even chemical damage
to anodes (Ferreira-Aparicio et al., 2009). Voltage reversal in fact is a well understood
phenomenon in electronic circuit and battery theories, linked to an imbalanced increase in
Rint of a cell that is part of a stack (Ieropoulos et al., 2010a). Although the reasons of Rint
increase can be varied, including electrolyte depletion or membrane fouling, the ultimate
cause for reversal is the increase of internal resistance and not substrate imbalance or
starvation per se. A thorough explanation particularly oriented to MFCs of this
phenomenon has been previously detailed by Greenman et al. (2011) .
Further research is needed to fully understand what the actual effects of starvation are on
MFC biofilms and stackability, but undoubtedly, starvation should not necessarily be
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
avoided. Selectively depriving MFCs could result in the necessary robustness to operate with
e.g. household wastewaters, where the presence of antimicrobial agents is ever increasing.
Conclusion
This investigation showcases MFC-Cascades as an uncomplicated and effective way to
reduce >96% COD from high-load effluents to acceptable environmental standards with
concomitant electricity production. In terms of MFC stacking, it is demonstrated that a
beneficial effect occurs in the form of superior performance (higher power and current
densities) when multiple units are combined together in comparison to single units,
provided correct conditions are set, particularly fluidic/electrical isolation when in series
configuration. The results also tackle the idea that voltage reversal, and thus unstable
performance, are an obligated occurrence when MFC stacks are operated in series or
starved.
Acknowledgements
The authors would like to thank Sam Coupland of the Bristol Robotics Laboratory for his help
in the design and fabrication of the MFC parts. Pablo Ledezma is supported by the National
Science and Technology Council of Mexico (CONACYT) Ref. 206298. Ioannis Ieropoulos is
supported by the Engineering and Physical Sciences Research Council of the UK (EPSRC) CAF
EP/I004653/1.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
Figures
Fig. 1. 3D CAD model of the stack; MFCs 1 to 6 are numbered according to their position in
the cascade.
4
Power density [W.m
-3
]
-3
-3
Cell MPD [W.m ] CCD [A.m ]
MFC1
3.69
14.32
MFC1
MFC2
3.23
13.39
MFC2
MFC3
3.12
13.16
MFC3
MFC4
2.87
10.43
MFC4
MFC5
2.47
7.67
MFC5
MFC6
2.19
7.22
MFC6
3
2
1
0
0
10
20
30
Current density [A.m
40
-3
50
]
Fig. 2. Polarisation experiment of 6 MFCs in cascade with a single feed at the top – individual
operation. Inset table: Maximum Power Density (MPD) points achieved during the
polarisation for each MFC (normalised); the number indicates the position on the cascade
(see Fig. 1). CCD: Corresponding Current Density (normalised) for the MPPs.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
2000
5mM Acetate + TYE 1
5mM Acetate + TYE 2
Artificial wastewater
64.1%
2
3
4
5
6
7
8
96%
1
95.8%
0
95.6%
EU WWTD 125
0
31.7%
500
95.3%
1000
88.4%
COD [mg.L -1]
1500
9
10
11
12
Number of MFCs in the cascade
Fig. 3. Combined COD reduction over number of MFCs for 5 mM Acetate TYE (average of
two independent repeats marked with  and  symbols) and artificial wastewater (avg. in
pink ). The dashed curves indicate the overall tendency in COD reduction. The horizontal
limit at 125 mg.L-1 (in green) marks the minimum acceptable levels of COD for release into
the environment according to the EU WWTD. Inset boxes indicate the amount of COD
reduction in percentage, relative to the original COD of the feed.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
Fig. 4. Different stack configurations (simplified to 2MFCs). a. Connections in parallel with
the original fluidic configuration: the flow of electrons (indicated by arrows) is concurrent
with the cross-conduction (2-way arrow) between fluidically-linked (see blue tube)
cathodes, resulting in no observable losses. b. Change to series configuration: electric
cables (black) now link the anode (bot. left; yellow) to the top cathode (top right; pink);
however, due to cross-conductance between cathodes (fluidic bridge in blue), some of the
electrons produced at the bottom anode end up at the bottom cathode (bot. right) without
passing through the external load (see red curved arrow), resulting in a direct short-circuit
or ‘shunt losses’ which negatively affect the performance measured at Rext . c. With the
introduction of fluidic insulation between cathodes (mirror image of the anode structure),
‘shunt’ losses are avoided and the stack exhibits a performance equivalent to the theoretical
prediction.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
10
6MFC stack in parallel
6MFC stack in series
8
Power density [W.m
-3
]
Individual MFCs (avg.)
6
4
2
0
0
20
40
60
Current density [A.m
80
100
-3
]
Fig. 5. Comparison of power curves between reference single MFC units and a 6MFC stack
connected in parallel or series configurations, showcasing the positive stacking effect.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
Current density [A.m
-3
]
20
15
A
B
C
10
5
0
0
200
400
600
800
Time [hours]
8
G
Power density [W.m
-3
]
F
6
4
E
D
2
0
0
100
200
300
400
Time [hours]
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
Fig. 6. Starvation cycles of a 6MFC cascade stack connected in a: parallel and b: series
configuration. All cycles are presented to scale, but were carried out during the several
months of testing (discontinuity is marked by ├─┤). : Start of a cycle (anolyte pump
off). : restart of the feeding (pump on). The length and chronological separation of the
cycles are as follows: A: 110.6 h; 3 weeks after joining the 6MFCs in parallel. B: 106.6 h; 5
weeks later. C: 355 h; 5 weeks later. D: 51.9 h; 1 week after connecting the stack with true
insulation between feeds. E: 78 h; 2 weeks later. F: 43 h; 10 weeks after E. G: 50.5 h; 4
weeks after F.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
Tables
Table 1.
a
b
Individual
6MFC Stack
Individual
6MFC Stack
MFCs (avg.)
MFCs (avg.)
MPP (µW)
120.9
17.3
268.36
40.99
Equiv. MPP per MFC (µW)
20.15
17.3
44.73
40.99
MPD (W.m-3)
3.35
2.88
7.46
6.83
MCP (mA)
1.39
0.17
2.63
0.41
Equiv. MCP per MFC (mA)
0.23
0.17
3.28
0.41
MCD (A.m-3)
38.61
28.33
91.11
85
Table captions
Table 1. Comparative results of polarisation sweeps between a 6MFC stack and single
individual cells. a: after one week in parallel configuration. b: after 8 months in parallel
mode. MPP: Maximum power point. MPD: Maximum power density. MCP: Maximum
current point. MPD: Maximum power density.
This is a post-print version of the following article: Ledezma, Pablo, Greenman, John and Ieropoulos,
Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119
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Ioannis (2013) MFC-cascade stacks maximise COD reduction and avoid voltage reversal under
adverse conditions. Bioresource Technology, 134 158-165. doi:10.1016/j.biortech.2013.01.119