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Maximising electricity production by controlling the biofilm specific growth rate in
Microbial Fuel Cells
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Pablo Ledezma1*, John Greenman2 and Ioannis Ieropoulos1
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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.
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* Corresponding author: pablo.ledezma@brl.ac.uk
Telephone: +44 (0) 117 32 86350
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Fax: +44 (0) 117 32 83960
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Abstract
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The aim of this work is to study the relationship between growth rate and electricity production in
perfusion-electrode Microbial Fuel Cells (MFCs), across a wide range of flow rates by comeasurement of electrical output and changes in population numbers by viable counts and optical
density. The experiments hereby presented demonstrate, for the first time to the authors’
knowledge, that the anodic biofilm specific growth rate can be determined and controlled in
common with other loose matrix perfusion systems. Feeding with nutrient-limiting conditions at a
critical flow rate (50.8 mL.h-1) resulted in the first experimental determination of maximum specific
growth rate μmax (19.8 .day-1) for Shewanella spp. MFC biofilms, which is considerably higher than
those predicted or assumed via mathematical modelling. It is also shown that, under carbon-energy
limiting conditions there is a strong direct relationship between growth rate and electrical power
output, with μmax coinciding with maximum electrical power production.
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Keywords
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Microbial fuel cells; Shewanella oneidensis; specific growth rate
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1. Introduction
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Microbial Fuel Cells (MFCs) are an exciting technology for the direct, sustainable recovery of
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electricity from different organic sources and notably organic wastes. Recently, considerable
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
research effort has been put into the composition and configuration of these systems, with
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numerous publications appearing frequently. However, it is not often reported that ultimately it is
the microbial cells, growing as a biofilm inside the fuel cell, that generate the electrons depending
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on their metabolic rates. Understanding growth and metabolism is key to controlling the reactions
that occur inside the system and dictate the performance, especially for practical applications. For
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example, in the case of MFCs for wastewater treatment, knowing the bacterial specific growth rate μ
is fundamental, as in any other biological treatment processes, for optimal calibration of the
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operational settings (Pollard & Greenfield, 1997). It has been said that a good wastewater treatment
plant has the inherent ability to exploit the growth of the bacterial population, so that it converts
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waste to products and biomass in the desired proportions (Henze, 2007). A similar reasoning can be
applied to MFCs: the right operational settings can lead to a desired proportion of biomass
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production and respiration via the anode; maximising the latter is of outmost importance for MFCs
employed as a bioelectrochemical power source.
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Helmstetter and Cummings (1963) first demonstrated that synchronous populations of bacteria,
based on dynamic steady-states, could be obtained from a biofilm; their work also showcased the
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possibility to precisely control the growth rate and physicochemical parameters in a similar way to
that achieved in a chemostat, providing that the attachment matrix allowed for homogeneous
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perfusion of fresh nutrient medium to all cells (Gilbert et al., 1989). MFCs based on carbon veil
electrodes have already demonstrated such dynamic steady states (Greenman et al., 2006). The
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present study describes, for the first time to the authors’ knowledge, measurements of cell
production and matrix-electrode biofilm growth rates, revealing a strong relationship between μ and
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electricity production in MFCs.
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2. Material and methods
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
2.1 MFC assembly and operation
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Eight standard 20mL/side two-chamber MFCs fabricated via rapid-prototyping (Ledezma et al., 2010)
with 270 cm2 plain carbon veil electrodes (Melhuish et al., 2006), were inoculated with S. oneidensis
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MR-1 and fed with modified M1 minimal medium based on Myers & Nealson (1988) with 18 mM
sodium-DL-lactate as the sole carbon source. The medium was fed continuously, initially at a low
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flow rate of 7.2 mL.h-1 (hereafter referred to as setting 1) until steady state electrode readings were
obtained and perfusion concentrations of daughter cells (cfu.mL-1) were constant (approx. 3 weeks)
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(data not shown). Subsequently, the flow rate was changed to 14.5, 27.3, 50.8, 72.6 and 94.4 mL.h-1
(settings 2 to 6 respectively) and the effects on μ and power production were determined. The MFCs
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were fitted with an anti grow-back device placed at the inlet port to ensure that no degradation of
the substrate occurred prior entry to the anodic chambers. All experiments were conducted at room
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temperature 23 ± 2 °C.
2.2 Specific growth rate μ
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2.2.1. Production rate of daughter cells
Once steady states were reached, anolyte samples (2 mL) were collected from the MFCs’ outlet flow
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ports. A 1mL volume was used to determine optical density at 540 nm (OD540nm). The remaining
sample volume was used for triplicate viable counts (cfu.mL-1) by serial 10-fold dilution and spiral-
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plating (Thorn & Greenman, 2009); by multiplying the latter by the set flow rate (mL.h-1), the
production rate of daughter cells eluted into the spent perfusate (cfu.h-1) was determined.
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2.2.2. Biofilm population
The experimental steady-state settings and measurement phases were concluded after
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approximately 10 weeks, following which the MFCs were disassembled and the electrode biofilms
were extracted (Lovley & Phillips, 1988) and processed as in 2.2.1. The determined average number
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
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of cells.mL-1 was multiplied by the total volume of the extraction (40 mL PBS) to obtain the total
viable cell population per electrode-biofilm.
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2.2.3. Calculation of specific growth rate μ
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The specific growth rate μ of the MFC biofilms at each particular flow rate settings was calculated
using the following formula (Greenman et al., 2002):
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μ (.h-1) = production rate of cells in perfusate (cells.h-1)/biofilm population (total number of cells)
2.2.4. Theoretical framework
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In the presented MFC systems, the folded carbon-veil electrodes form a structure of 38x28x18.5 mm
inside each 20 cm3 chamber, leaving very little void volume available for planktonic growth. The
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former model is thus based on the assumption that once the loose matrix porous electrode is fully
colonised, all suitable attachment sites for electron respiration are saturated. In continuous flow,
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attached cells exist in a non-cumulative steady state, maintaining a near-constant number
(Greenman et al., 2006) in a monolayer or thin film form (Helmstetter & Cummings, 1963), while
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new cells detach into the perfusate flow. Due to the permeability of carbon veil electrodes, cells are
uniformly supplied with rate-limiting nutrients, continuously growing at approximately the same
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rate, and responding equally to shifts in physicochemical conditions (e.g. changes in hydrodynamic
flow), with direct consequences for electricity production. This is in opposition to thick biofilms on
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impermeable surfaces, where it has been demonstrated that substrate diffusion is reduced, creating
gradients that lead to differential metabolic activity and thus a wide distribution of μ depending on
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the cell’s location within the biofilm (Picioreanu et al., 2007); thin biofilms do not exhibit such
behaviour and have already been demonstrated for Shewanella oneidensis (Bretschger et al., 2007).
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It is also assumed that electron transfer from biofilm to electrode is by conductance rather than by
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
soluble redox mediators found in the bulk liquid. With the employed flow rates, the Hydraulic
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Retention Times (HRTs) range from 2.29 to 0.17 h; at moderate and high replacement rates (more
than 5 times per hour for setting 6), any mediators would be expected to diffuse and be removed,
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thus playing no significant role in electricity production over reasonable amounts of time (>10
minutes).
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2.3 Electrical output
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MFC voltage output with a 2.2 kΩ external resistor was recorded and the electric power production
(in μW) was calculated (Ieropoulos et al., 2008); the load was selected for maximum power
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production by impedance matching, based on repeated polarisation tests of the MFCs (data not
shown).
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3. Results & Discussion
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3.1 Effects of flow rate on specific growth rate
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For non-MFC perfusion biofilm systems in steady state and carbon-energy (C/E) nutrient-limiting
conditions, an increase in supply rate has been demonstrated to cause a proportional increase in μ,
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up to a critical flow rate (CFR), where maximum specific growth rate (μmax) is achieved (Gilbert et al.,
1989). Results (Fig. 1) indicate that MFC systems can also exhibit such behaviour. Steady states are
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demonstrated by constant power output e.g. 42.29 ± 0.06 μW over 8 hours under setting 3,
indicative of a stable metabolic activity (see Inset graph, Fig. 1). Separated effluent samplings (see A,
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B & C in Inset graph, Fig. 1) exhibited near-identical OD540nm (0.563 ± 0.002, 0.561 ± 0.003 and 0.560
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
± 0.002 respectively; ¼ PBS dilution in all cases). Viable counts then confirmed that the number of
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cells in the perfusate was stable (A: 1.77 ± 0.14 x 109; C: 1.76 ± 0.15 x 109 cfu.mL-1) while the biofilm
extractions performed later on revealed that the populations attached to the electrodes were in the
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order of ~1011 for all MFCs. The latter evidence confirms the theoretical framework presented in
2.2.4 whereby the grand majority of the cells in the system are attached and respire directly and
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stably to the electrode, while the electrical output of the planktonic cells (<2% of total cell
population in the system, constantly removed by the perfusion flow) by soluble mediators or other
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means is negligible particularly over large periods of time such as those presented in Fig. 1 Inset.
Moreover, a quasi-linear increase in μ up to a CFR (50.82 mL.h-1) was observed in proportion to flow
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rate (r2= 0.988; p<0.0005). Further increasing the supply rate resulted in no faster growth (see
settings 5 and 6, Fig.1); thus, for the particular conditions of these experiments, a μmax of 0.827 ±
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0.004 .h-1 was reached. To the authors’ knowledge, there are only two previous articles where μmax is
reported for bioelectrochemical systems (Lee et al., 2009; Pinto et al., 2010), though both are
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mathematical estimations and not directly measured or based on real experiments. The biofilm μmax
obtained for Shewanella (19.85 .day-1) is 6- or 7-fold higher than calculated in cited papers (3.2 .day-1
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and 1.97 .day-1 respectively). Results demonstrate accordingly that the specific growth rate of the
biofilm inside a MFC can be controlled and maximised (within the limits set by the CFR), as in other
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perfusion systems, to previously unpredicted levels.
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3.2 Effects of flow and specific growth rate μ on Power production
As depicted in Fig. 2A, an increased flow rate gives a rise in power production up to a limit (a
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phenomenon also observed by Venkata Mohan et al. (2007) and Behera & Ghangrekar (2009)),
which coincides with the CFR for μmax (see setting 4, Fig. 1). Therefore, maximum continuous power
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
150
production (Pmax) was recorded when the system was configured to achieve μmax. Steady state
outputs were achieved at higher flow rates (and found to be reversible i.e. same power output when
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the flow-rate was decreased to a previous setting), confirming that the biofilm layers remained
attached up to circa 95 mL.h-1 (further research is needed to determine whether shear forces alone
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are sufficient to remove daughter cells or other shedding mechanisms are involved).
Though μ is not synonymous with metabolic rate, the former is ultimately controlled by the latter; at
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faster growth rates, proton pumping through the lipid membrane would be expected to increase; in
the specific case of MFCs, this results in higher electricity production. This positive relationship is
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depicted in Fig. 2B (fit: r2=0.954; p<0.0005), although confined by the “boundaries” imposed by μmax
and Pmax. Flow rates higher than the CFR achieve neither higher power nor growth; this could be
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explained by: (i) more nutrients passed through the anodic chamber without reaction; (ii) a faster
removal of produced H+ (flowing out instead of passing through the PEM); and (iii) Shewanella could
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not grow any faster under these conditions. It should be noted that said limits are experimentspecific; many other MFC systems exhibit higher electricity production (e.g. with the help of
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catalysts, non-limiting conditions), but these would still be restricted by μmax. As previously
mentioned, determining μ is essential for wastewater treatment systems; the obtained results
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demonstrate that, under nutrient-limiting conditions, μmax brings about sustainable Pmax. Such
mechanisms could therefore be utilised to achieve maximum specific growth rates, for both
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maximum electricity production and transformation rates of C, N, P, K, Mg (among others) into new
biomass. Further research is now needed to determine the behaviour of the system under C/E
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excess (i.e. limited by a different class of nutrient).
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Conclusions
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
The experiments presented demonstrate that the specific growth rate of bacterial biofilms inside a
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MFC can be controlled. It is also shown that under C/E-limiting conditions, maximum growth results
in maximum power production, with μmax considerably higher than previously estimated. It has been
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suggested that knowing the growth kinetics of Shewanella oneidensis MR-1 is essential to fully
explore its potential in fuel cells (Tang et al., 2007); this is clearly validated here, calling for similar
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studies for other anodophile species, MFC designs, thick biofilms and conditions other than C/Elimitation so that MFCs can be practically used as bioelectrochemical power sources.
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Acknowledgements
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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.
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This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
Figures
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Fig. 1. Relationship between flow rate and specific growth rate μ. For data below the CFR, the linear
fit is presented. Inset graph: Example of steady power production under flow setting 3.
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
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Fig. 2. A: Average power production of MFCs vs perfusate flow rate. B: Average Power production as
a function of specific growth rate. Experimental flow rate settings are indicated with numbers (1 to
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6).
This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008
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This is a post-print version of the following article: Ledezma, Pablo, Degrenne, Nicolas, Bevilacqua,
Pascal, Buret, François, Allard, Bruno, Greenman, John and Ieropoulos, Ioannis (2014) Dynamic
polarisation reveals differential steady-state stabilisation and capacitive-like behaviour in microbial
fuel cells. Sustainable Energy Technologies and Assessments, 5 1-6. doi:10.1016/j.seta.2013.10.008