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Relation of anodic and cathodic performance to pH variationsin membraneless microbial fuel cells

Edoardo Guerrini b,1, Pierangela Cristiani a,*, Stefano Pierpaolo Marcello Trasatti b,1

aRSE e Ricerca sul Sistema Energetico S.p.A, Environmental and Sustainable Development Department, V. Rubattino 54, 20133 Milan, ItalybUniversita degli Studi di Milano, Department of Physical Chemistry and Electrochemistry, Via Golgi 19, 20133 Milan, Italy

a r t i c l e i n f o

Article history:

Received 15 March 2012

Received in revised form

24 September 2012

Accepted 1 October 2012

Available online 27 October 2012

Keywords:

Pt-free cathode

Biocathode

Sulfur acetate Pourbaix diagram

Membraneless microbial fuel cell

pH variation

* Corresponding author.E-mail addresses: edoardo.guerrini@unim

1 Tel.: þ39 02 3992 4655.0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2012.10.0

a b s t r a c t

One-compartment (membraneless) microbial fuel cells (MFCs) are effective tools to test

new bio-technology at a laboratory level. More efforts in MFC design and materials are

necessary to move from laboratory tests to real applications.

In such a context, this paper presents the experimental results that investigate pH

variations of three single chamber and membraneless MFCs having positive and negative

electrodes made of graphite-based materials without any addition of chemical catalysts.

MFCs were built and operated with raw wastewater (inoculum) and sodium acetate as

substrate. The progression of the power in the MFC and the relationship between perfor-

mances and induced pH variation (from pH 6.7 to 10.2) will be discussed. A general

connection between SEM images, chemical analyses, pH trends and reactions in the MFCs

will be attempted, by connecting all processes with thermodynamic and chemical equi-

libria considerations.

Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction substances into carbon dioxide, hydrogen-ions and electrons,

A microbial fuel cell (MFC) is an innovative electrochemical

bio-technology to produce electricity directly from wet

marginal biomass and wastes. Studies have boosted in the

end of the last century, mostly due to the discovery of the

capability of different bacteria to catalyze oxidation reactions

and to transfer electrons directly in conductive metals

(anodes) [1]. On the other hand, the study of corrosion induced

by electrochemically active biofilms (Microbial Induced

Corrosion) goes back to the beginning of the last century [2].

MFC technology aims to physically separate the oxidation

from the reduction process (as all batteries and fuel cells),

thus forcing bacteria to use electrodes as intermediates in the

electron cascade process [1]. The bacteria grow on the anode

and form a biofilm able to carry out the oxidation of organic

i.it (E. Guerrini), Pierange

2012, Hydrogen Energy P01

in the absence of oxygen. Interest on MFCs is continuously

increasing [3e5]. The reason can be found on the simplicity of

the principal constituents. In particular, the possibility of

using industrial, agricultural, or anthropogenic organic

wastes as energy vectors could open new ways to achieve

distributed electrical power for emerging countries. A broad

range of potential MFC applications is in wastewater treat-

ment, where the biodegradation processes are crucial and

require significant consumption of electricity [6]. Despite the

attractive feature of power production with wastes and

a cheap apparatus (if compared with other fuel cells) this

technology needs a deeper study of the involved mechanisms

[7] and is still far from being applied at an industrial level,

even though a few attempts for the scaling-up process can be

found [8].

la.Cristiani@rse-web.it (P. Cristiani).

ublications, LLC. Published by Elsevier Ltd. All rights reserved.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 4 5e3 5 3346

More efforts in MFC design and materials are still neces-

sary to move from laboratory tests to real applications,

including use of cheap materials and improvement of long-

term stability of anodic and cathodic processes.

The first flywheel of this way of thinking is the possibility

to eliminate ionic (protonic) membranes, because of their

costs and their ordinary tendency to clog if exposed to

microbial suspensions. Similar issues relate to platinum, the

usual/best electrocatalyst used for the chemical oxygen

reduction reaction (ORR). From Direct Methanol Fuel Cells

(DMFCs) technology, it is well known that platinum-based

catalysts get poisoned by the by-products of the oxidation of

organic substances, like carbon monoxide (CO) [9]. CO is the

main poison for pure-platinum catalysts, even if dispersed in

a highly porous conductive matrix like carbon powder [10]. A

few ppb of methanol in the cathodic compartment could

prevent Pt from being an ORR electrocatalyst and many

studies dealt with this problem in the past [10e12]. As a result,

involvement of simple Pt catalysts in the oxidation of organic

substances will cause a more or less quick deactivation of its

electrocatalytic power, and several studies already demon-

strated similar performances of platinum-free graphite cath-

odes [13,14].

In such a context, experiments have been carried out and

are still in progress by means of very simple bio-

electrochemical systems, made of membraneless, single

chamber MFCs (SCMFCs). Electrodes are made of graphite-

based materials in the absence of platinum and any other

chemical catalyst [15,16].

This paper will attempt to show, and possibly explain,

experimental observations about the influence of pH on the

cathodic and anodic processes involved in the power gener-

ation of SCMFCs fed with sodium acetate, which is the

simplest, final organic product of bacteria catabolism [3].

As a simple statement, pH is the result of proton balance in

all chemical and biochemical reactions in the MFC. Here, the

two half-reactions in our MFCs will be briefly recalled:

The final cathodic reaction is the reduction of molecular

oxygen to water (ORR):

2O2 þ 8e� þ 8Hþ / 4H2O (1)

The anodic reaction is the oxidation of sodium acetate, fed

to the MFC, to CO2. Seven protons are released during this

process:

CH3COO� þ 2H2O / 2CO2 þ 8e� þ 7Hþ (2)

It is worth noting the difference in the protons balance,

with equivalent electron exchange, that promotes the pH

modification in time. MFC geometry and subdivision plays

a key role in the determination and distribution of the pH in

the MFC.

Discussion of the results will be based on the pH trends (vs.

time), and on the current generation trends, taking into

account the coulombic efficiencies exhibited by the MFCs.

Data are supported by optical and SEM images and EDX

analyses.

2. Materials

TheMFCswere operating at 30� 3 �C (Fig. 1) with an inoculum

from raw wastewater and a single initial substrate addition of

3 g L�1 of sodium acetate. MFCs described in this paper are

a sub-set of 20 MFCs with the simple laboratory configuration

(although not the most efficient) shown in Fig. 1, with exactly

the same volume, anode, wastewater and MFC design.

Differentiation has been made by furnishing the MFCs with

different cathodes. Because of the subject of this paper, only

three MFCs will be presented.

All MFCs used in this experimentation were constituted of

a glass single chamber (SCMFC) (Fig. 1). Anodes were put on

the bottom of the MFC by one of the two lateral inlets. Oppo-

site to these two inlets, a bigger flange held in place the

cathodes. At the top of the MFC, a screw-cap was used to

prevent oxygen exchange between solution and air.

Anodes were made of carbon cloth (5 cm2 geometric area).

No particular treatments were made to the anodes, and new

carbon cloth was used as-received (from Saati, Legnano Italy).

Cathodes were a multilayer made in two different ways.

Conductivity of electrodes was always assured by a carbon

cloth sheet (5 cm2 geometric area). A first kind of cathodes

(MFCs No. 15 and 16) was built adding an inner layer of

graphite-based (TIMCAL, 250 mmdiameter) gas diffusion layer,

as previously described [16]. The second kind (MFC No. 18) of

cathodes wasmade in the samemanner, but using a graphite-

based powder of 350 mm diameter (TIMCAL). Both kinds were

produced without any catalyst. So, from the practical point of

view, cathodes were all built with the same components,

varying only the diameter of the carbon conductive powder.

Raw not-diluted wastewater, from Nosedo depuration

plant in Milan, was used as inoculum of bacteria consortium

for all MFCs. No electrolyte or pH buffers were added.

Conductivity of wastewater was measured and resulted to be

w1 mS cm�1. Modifications of chemical composition were

made only by periodically adding (on a weekly base) 3 g L�1 of

sodium acetate.

3. Methods

During all the experiments, the cathode was electrically con-

nected to the anode by an external resistor (R) of 100 U. Based

on previous experiments, this value of the external load was

chosen because it approached the maximum of the power

curves. The corresponding potential difference (E ) across the

resistor was recorded every 15 min, by means of a multi-

channel potentiometer (Graphtech midi Logger820) for each

MFC. Current production (I ) is then directly related to voltage

by the ohmic law (E ¼ RI ).

pH was measured each week (Amel pH-meter, model 337).

Standardization of the pH glass-electrode was made each

morning, and when necessary, with pH 7 and 9 buffers.

Titration of wastewater was performed to assess the capa-

bility of natural wastewater to buffer pH variations eventually

induced by bacteria.

Chemical Oxygen Demand (COD) is the parameter used to

measure (in terms of grams of O2) the substances oxidizable

Fig. 1 e Image and sketch of a single chamber membraneless MFC.

i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 4 5e3 5 3 347

by an excess of chromic acid in the MFCs. Carlo Erba instru-

mentation and methods (IDRIMETER, range:100e1500 or

1000e5000 mg L�1) were used.

Dried samples of electrodes were analyzed by means of

Scanning Electron Microscope and the surfaces were chemi-

cally characterized by EDX microprobe.

Many physico-chemical and biological parameters

acquired during the experimentation (e.g.: anodic, cathodic,

open circuit, short circuit potentials; COD trends; bacteria

molecular analysis; conductivity trends) are reported else-

where [15].

3.1. Coulombic efficiency

By the comparison of the current levels exhibited by eachMFC

and the COD variation during the same period, the Coulombic

Efficiency (CE) was calculated. The coulombic charge passed

through the external 100 U load was estimated by trapezoidal

integration of the current-time trends. The time interval was

chosen to fit the period between each acetate addition.

Calculation of average CE, was performed.

3.2. Polarization curves

In order to distinguish anodic from cathodic behavior and

contribution, polarization curves were acquired with a typical

three-electrodes configuration (working, counter, and reference).

The working was the electrode to be tested: respectively,

cathode or anode for the cathodic or anodic polarization

curves. Counter electrode was a Pt wire (0.3 mm diameter,

100 mm long). For all experiments here reported, referencewas

SCE (Standard Calomel Electrode, þ0.242 mV vs. RHE). For the

anodic curves, the reference electrode was put as near as

possible to the anode. For cathodic curves, a glass Luggin

capillary holding the reference was put with the opening of

the capillary close to the cathode. Thus, in both cases, the

reference electrode was set so as to minimize ohmic losses

due to solution and electrolyte resistances. Briefly, during

a typical polarization curve, the systemwas left at open circuit

for 10 min, to stabilize the open-circuit-potential. The poten-

tial was then drifted at a speed of 10 mV min�1 in anodic or

cathodic directions, as needed. One point (mV e Current) was

automatically recorded each minute. The potential scan was

stopped when an absolute current of 10 mA was reached.

3.3. Thermodynamic data derivation

Thermodynamic data, mostly DfG0 of each investigated

compound, was taken from literature [21]. To calculate

chemical equilibria, like acid dissociation constants, the

investigated reaction was written and DrG0 was determined.

Then, with the usual relation DrG0¼�R$T$ln(K ), the particular

equilibrium constant was calculated, where R is the universal

gas constant, T the temperature in Kelvin, and K the target

equilibrium constant. Calculation of standard electrochemical

reduction potentials (DE0) was performed determining the

reduction half-reaction DrG0 in the same manner, and

applying the relation DrG0 ¼ �n$F$DE0, where n is the number

of electrons and F the Faraday constant. To obtain the slope of

electrochemical reactions in the Pourbaix diagram, the Nernst

equation was applied, assuming unity for the concentrations

of both reagents and products, except Hþ.

4. Results and discussion

4.1. Wastewater titration

From previous analyses, wastewater from the Nosedo depu-

rator in Milan contains the residue of organic compounds

testified by an initial COD of 0.5 g L�1. Keeping inmind the aim

of this study, an acid-base titration of 50 mL of wastewater

was performed. The corresponding titration curve is reported

in Fig. 2.

As shown in Fig. 2, the initial pH of the wastewater was

about 7.8. It is important here to say that initial pH depends

on the wastewater samples and changes during aging. In any

case, bacteria films developed on the electrodes. Titration

was performed to give an indication of the capability of the

wastewater to minimize and smooth pH potential changes

during the running of the MFCs. In the caustic region (posi-

tive abscissa values) a slow and continuous increase in pH is

Fig. 2 e pH variation during a titration of the raw

wastewater with HCl 0.01 M (Negative values) and NaOH

0.01 M (positive values).

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 4 5e3 5 3348

noticed as NaOH is added. This slow increase of pH is thus

explainable by an effective buffer capacity of the wastewater

toward caustic matter addition. A mathematical number can

be extracted by Fig. 2: interpolation of the points on the right

Fig. 3 e Images of cathode surfaces. Water side surface: (a) SEM

Air-side surface: (b) SEM at 5003 and insert of entire electrode

part, from 0 to 20 mL (linear part of the graph) gives a slope

of 0.09 pH Units mL�1 of NaOH 0.01 M. The negative region of

the graph in Fig. 2 shows the behavior of wastewater toward

HCl 0.01 M addition. Here, the buffer capability is less

effective, as after about 15 mL HCl addition a sudden

decrease in pH takes place. As a consequence of these two

additions, a general deduction can be drawn: raw waste-

water possesses discrete buffer capability, especially visible

for alkaline drifts.

4.2. Cathode images and analysis

Fig. 3 shows images of the opposing sides of the cathodic

electrode, exposed to oxygen. A thick biofilm formed on the

inner water-side surface (Fig. 3a and insert) of the carbon

electrode for all types of cathodes. On the outer air-exposed

surface, biofilm and salts precipitation were also evident

(Fig. 3b and insert). EDX analysis (Fig. 3c and d) indicates

predominant CaCO3 on the inside and Na2CO3 on the outside

the cathodes [17]. Other organic salts can be present, but the

carbon signal was saturated by the graphitic nature of the

electrode.

at 5003 and insert of entire electrode area; (c) EDX analysis.

area; (d) EDX analysis.

i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 4 5e3 5 3 349

The presence of sulfur based compounds was revealed by

EDX, especially on the water side. This aspect will be dis-

cussed in Section 4.5.

Bacteria colonization is clearly evident. Bacterial growth

covers electrodic surfaces, and the space between carbon

fibers as well. Carbonate deposits are visible all over the

surface, recognizable by the whiter round flakes in Fig. 3a. A

thick biofilm, mixed with carbonate deposits, could act as

organic/inorganic membrane, modifying the diffusion coeffi-

cients of species in the solution, in particular acetate and O2.

When acetate has limited access to the cathode, Coulombic

Efficiency is expected to increase. Oxygen diffusion limitation

acts in the same way, limiting the number of acetate ions

being catabolized directly to CO2 without the release of elec-

trons in the anode. In other words, oxygen diffusion limitation

helps keep the electrolyte anaerobic. At the same time, biofilm

metabolism can reduce oxygen coming from air, helping once

more to keep anaerobic conditions in the rest of the solution.

As reported in a previous work [18], the biofilm of the cathode

was colonized mostly by Sulfate Reducing Bacteria (SRB) and

other anaerobic species. These bacteria can survive in the

presence of low oxygen concentrations, and use oxygen as

terminal electron acceptor, even though growth stops. High

oxygen concentrations can cause SRB migration to the oxic/

anoxic zone. Successive Oxygen respiration by the outer

regions of biofilm stimulates SRBmetabolism reactivation [19]

inside the biofilm. This is consistent with the biological

analysis on our cathodes and with the thick biofilm on the

water-side. Amore comprehensive and exhaustive discussion

of the possible role of the different bacteria will be the topic of

forthcoming more targeted research.

4.3. pH vs. potential trends

Average Coulombic Efficiencies were calculated after three

weeks, and resulted to be as high as w50% for all three MFCs

(cell 15 ¼ 44%, cell 16 ¼ 47%, cell 18 ¼ 54%). Fig. 4 shows the

variation of voltage trends (at 100 U load) of the three MFCs.

Electrode colonization takes less than one week to stabilize.

During this period, voltage grows up to its maximum and then

reaches a plateau.While the different plateau of the two types

Fig. 4 e Voltage trends (left axis) and pH trends (right axis)

at the beginning of the experimentation, for the three

carbon-based cells.

of MFCs could be barely ascribed to cathode porosity differ-

ences, pH trends show a more clear correlation.

As shown in Fig. 4, pH decreases in all MFCs at the very

beginning of the experimentation (two days).

This can be due to the process of fermentation, whose final

result is the production of an anaerobic environment inside

the MFC [20]. It is interesting that this decrease is more

marked in the MFC No. 18 with carbon powder of 350 mm in

diameter. As the solution gets anaerobic, MFCs start to

produce electricity, and colonization of electrodes takes place.

Here again, pH shows a particular behavior linked with

voltage modifications. The lowest pH values related with the

highest voltages. So the MFC No. 18 exhibits voltages of 60 mV

and the pH remains constant at the lowest values.

This evidence is valid also for the carbon-250 mm equipped

MFCs No. 15 and 16. Even if voltage trends are quite similar for

these MFCs, some differences can still be found. First, at the

very beginning, MFC number 15 exhibits the highest pH, and

its voltage response is lower than MFC n. 16. In the same way,

on the right part of the graph, this small difference in voltage

becomes again visible, and pH goes parallel, as the difference

between the pH level for the two MFCs increases.

If the only reactions happening in the system are the ones

mentioned in Eqs. (1) and (2), a gradual accumulation of

hydroxyls should accompany acetate oxidation and oxygen

reduction. As a result, pH should gradually increasewith time,

becoming more alkaline as more acetate is removed. Thus,

MFCs with higher voltages (at equal CE) would cause a bigger

increase in pH. This is not the case, as shown by Fig. 4, so other

concomitant mechanisms must be involved in pH

determination.

A possible chemical explanation for these pH trends can be

argued, noting the following chemical reactions:

CO2 þ H2O / H2CO3 (3)

H2CO3/2 Hþ þ CO�23 (4)

CO�23 þ Caþ2/CaCO3 (5a)

CO�23 þ 2 Naþ/Na2CO3 (5b)

Concomitant reactions in Eqs. (5a) and (5b) show the

formation of the precipitates found with the EDX analysis. As

can be easily stated by solubility products of the two carbon-

ates, the first to precipitate is the calcium carbonate, followed

by the more soluble sodium carbonate. If the concentration of

carbonates and cations would be the only driving force of the

precipitation, more concentrate solutions would be found in

the outer face of the cathode (air exposed) because of the

easier evaporation of water.

A second mechanism forcing these reactions to happen, is

strictly connected with oxygen reduction (Eq. (1)). According

to Eq. (1), oxygen reduction to water uses 8 protons (or

equivalently forms 8 hydroxyls) making the pH near the

cathode more alkaline than the rest of the solution. As

a consequence of the local pH increase, Eqs. (3) and (4) are

forced to the right (Le Chatelier’s principle) and achievement

of the solubility limit conditions is improved.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 4 5e3 5 3350

The final result of carbonates precipitation is the produc-

tion of acidity by Eq. (4). The equilibrium between alkali

production and carbonates precipitation is thus a matter of

local alkalinity, possibility of water evaporation, and obvi-

ously presence of cations. Predominant oxygen reduction,

without carbonates precipitation and water evaporation (on

the cathode surface) causes an increase of alkalinity. If instead

carbonates can precipitate, increase of pH would not be so

large. It is worth noticing that each acetatemolecule produces

2 CO2 with a net increase of 1 OH�. If both CO2 would

precipitate as carbonates, a production of 4 Hþ would be ex-

pected. As a consequence, a decrease in pH is possible.

The possibility of carbonate precipitation inside the

cathode pores cannot be in any case excluded, causing

chemical clogging of the oxygen penetration pathways.

Further analyses have to be performed in order to clarify this

process.

4.4. Polarization curves

Polarization curves were recorded using a Luggin capillary for

the reference electrode (SCE), to physically eliminate ohmic

drops caused by solution resistance between the tested elec-

trode and the reference electrode. A modified version of the

Luggin capillary was built to fit our purposes in our apparatus.

A first attempt to correct potentials of polarization curves was

made by a method called “derivative method”, reported else-

where [21]. Calculations of ohmic drops resulted in less than

8 U (typical of carbon cloth). Fig. 5 shows cathodic (absolute

value of current) and anodic curves at three pH levels, without

ohmic drop correction.

As already stated, pH plays a key role in the behavior of the

MFCs, like in the initial and final current and voltage genera-

tion. In Fig. 5, a better understanding of this process from an

electrochemical point of view is shown. Here, pH was modi-

fied by addition of a solution of NaOH to theMFCs in the end of

the experimentation, up to the desired pH value, measured in

the bulk wastewater. As a result, the electrode was signifi-

cantly modified. Intersection of the curves with y-axis gives

the half-cell open circuit potential (ocp). Thus the difference

between anodic and cathodic ocp gives the open circuit

Fig. 5 e MPL cathode equipped cell. Cathodic and anodic

polarization curves at three different pH. 6.7: solid lines,

8.7: dashed lines and 10.2: dotted lines.

potential of the MFCs at the considered pH. Ocp of the MFCs

decreases as pH is increased.

Observation of cathodic and anodic trends are described

below:

1) anodes (curves starting at zero current and going upwards)

are significantly influenced by pH increase. The starting

potential (ocp) jumps upwards, as illustrated in case of pH

8.7. In case of pH 10.2, anodic polarization was shown to

start at the same potential as pH 8.7. After the startup of the

polarizations, a comparable diversification happens. An

oxidation diffusion-limited current plateau (vertical parts

of the graph) is noticed on the more acidic pH, which

decreases to lower current levels as pH is increased.

Moreover, as pH gets more caustic, a tail of incipient

massive (not-diffusion limited) oxidation takes place

(experiments concerning the chemicals involved in this

initial anodic oxidation are in progress).

When polarization was performed on the anode unmodified

by any NaOH addition, the pH was 6.7 and the biofilm was

already established on the carbon cloth. As a result, a peak of

anodic current is observed at the beginning of the curve (solid

line). Two consecutive polarization curves were performed on

this electrode, in order to verify and reproduce this peak. The

peak may be a result of the oxidation of byproducts, or inter-

mediate redox couples, involved in the colonization of the

electrode and transfer of electrons. Interesting, this peak

shows up at a potential of about �350 mV (SCE), thus at about

�590mVvs. RHE, a potential inwhich cytochromes are known

to act as electron-shuttles [7,22]. When pH was modified by

NaOH addition, this peak disappeared.

2) Cathodes (curves starting from zero current and going

downwards) show a behavior similar to anodes. Ocp moves

upwards as pH is increased, but in a smoother manner if

compared with the anodic ocp jump. No other particular

features can be seen from these cathodic polarizations.

Looking at the relationship between anode and cathode at

a single pH, in Fig. 5 it is evident that anode comes to a diffu-

sion limitation before the intersection with cathode, whose

polarization can be approximated as a straight line (with no

ohmic resistance as a Luggin capillary was used). This obser-

vation ismore evident as pH is increased. Consequently, MFCs

under investigation (at least with a carbon-basedmicroporous

cathode) are probably under anodic control despite the

absence of platinum for the electrocatalysis of the cathodic

ORR. The alkaline pH strengthens this case.

4.5. SRB role: a chemical thermodynamic point of view

This paragraph deals with thermodynamic considerations

about the reactions that are probably involved in this study.

Thermodynamic data was taken from the literature [23].

As already mentioned, pH changes have to be connected

with a mix of biological activity and concomitant or subse-

quent chemical reactions. While in literature the reduction

of two oxygen molecules to water is unambiguously

producing 8 hydroxyls, the effective oxidation of the acetate

ion is sometimes modified to explain directly the pH varia-

tions on a “case by case” basis. So in the introduction of this

i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 4 5e3 5 3 351

paper, acetate was oxidized up to CO2. Then, in paragraph

4.3, equilibrium between CO2 and carbonate was claimed to

assess the stable pH in the MFCs, if carbonate deposits are

present. In fact, not only carbonates can be formed, but

bicarbonates can also be responsible for the pH variations. At

the same time, if acetic acid (instead of acetate ion) was

used, a production of 8 protons would not change the pH of

the total redox reaction. Therefore, it still remains unknown

at which pH-range equilibria change and furthermore, what

are the consequent Standard reduction potential (E0)

variations.

In addition, EDX analyses proved the existence of sulfur

inside the cathodic electrode, and microbiological analyses

found Sulfate Reducing Bacteria [18]. It is well known that SRB

can catalyze and take advantage of all the steps of the sulfur

cycle [19], from reduction to re-oxidation of sulfate. A final

interesting related outcome is represented in Fig. 6, as

a Pourbaix diagram, where thermodynamic calculations are

extended to sulfate/sulfide chemistry and equilibria.

The reactions of interest in this context are chemical and

electrochemical. In Fig. 6, vertical lines represent a chemical

equilibrium of protonation/deprotonation; oblique lines

represent the E0 of the redox couple located over and under

the line. Some chemical compounds have been discarded due

to their marginal contribution at pH suitable for microbial

life.

In Fig. 6, the dotted line represents the thermodynamic

potential for hydrogen reduction. The slope of the dotted line

is equal to unity (59 mV/pH unit at 25 �C). The dashed line is

composed of two segments: before pH 4.75, acetic acid

concentration prevails on acetate. The corresponding reaction

is a modification of Eq. (2):

CH3COOH þ 2H2O / 2CO2 þ 8e� þ 8Hþ (6)

After pH 4.75, acetate prevails and the reaction is repre-

sented by Eq. (2). At the same time, the slope of the Nernst

equation decreases from unity (59 mV/pH unit) before acetic

acid pK, to 7/8 of unity (51.6 mV). Similar evaluations can be

seen for the E0/pH segments of sulfur. Here chemical equi-

libria getmore complicated by the possibilities of H2S, HS� and

Fig. 6 e Pourbaix diagram showing behavior of sulfate/

sulfide redox couple (solid line), superimposed with CO2/

acetate (dashed line) and HD/H2 (dotted line) redox couples.

S�2 for the reduced species, and HSO4� or SO4

�2 for the oxidized

ones. Vertical dashed lines divide the graph and show the

existence interval for all species. So, before pH 1.9, the reac-

tion of sulfate reduction is:

HSO�4 þ 8e� þ 9Hþ/H2Sþ 4H2O slope ¼ 9=8 (7)

From pH 1.9 to 7.04, the half-reaction is:

SO�24 þ 8e� þ 10Hþ/H2Sþ 4H2O slope ¼ 10=8 (8)

From pH 7.04 to 11.92 the half-reaction is:

SO�24 þ 8e� þ 9Hþ/HS� þ 4H2O slope ¼ 9=8 (9)

Finally, at pH higher than 11.92, the half-reaction is:

SO�24 þ 8e� þ 9Hþ/S�2 þ 4H2O slope ¼ 1 (10)

Before any deduction based on Fig. 6, it is necessary to

remember that thermodynamic data are determined in pure

water solutions, with all concentrations except [Hþ] equal to1. So the Pourbaix diagrams are only a hint of what may

be happening and what is prohibited. Another question

deals with the chemical deprotonation equilibria: a straight

vertical line divides different chemicals at the same oxi-

dation state, but at this boundary, real potential has a

smoother transition, because of the presence of both che-

micals. Finally, useful constants and parameters were

calculated using the thermodynamic data coming from a

single source, because of the large numbers of articles

present in literature.

Looking at Fig. 6, apart fromnumbers, a few considerations

can be made. First, acetate couple lays over hydrogen couple,

so direct reduction of hydrogen by acetate oxidation is ther-

modynamically unfavorable. An attempt of using bacteria to

produce hydrogen must thus imply the use of a further

polarization of the cell, at least represented by the vertical

distance from the two couples [24]. In this case, hydrogen

production is less demanding at an acidic pH.

The sulfur redox behavior is more complicated. Because

of the pH ranges exhibited by our MFCs, from neutral to pH

10, the thermodynamic sulfate reduction is represented by

Eq. (9). Energy gain from the use of acetate to reduce sulfate

is quite low, at these pH. A bigger energy gain could be

achieved by using H2 as an electron source, as it is indeed

the case if hydrogen would be present in the environment

[25]. An interesting deduction comes by looking at the

crossing of the sulfur graph with the acetate graph at about

pH 12. The thermodynamics predict that SRB can’t use

acetate as an electron-donor at very high pH, because

oxidation of acetate and reduction of sulfate is energetically

unfavorable. As a consequence, pH 12 should prevent SRB

metabolism, as it is suggested in the literature for H2S

emission control [26].

This predicted behavior is evident in the data in Fig. 5, in

which a sudden decay of voltage followed NaOH addition,

while pH reached a value of 10.2. This MFC didn’t recover in

one month, and showed a small voltage slowly increasing as

pH slowly decreased, caused most probably by air-induced

carbonatation of NaOH in the MFC. At the same time, the

MFC brought to pH 8.7 recovered slowly in time (data not

shown).

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 4 5e3 5 3352

5. Conclusions

MFCs represent a promising technology from many different

points of view. The use of bio-cathodes and the simplification

of MFC construction can boost their development and scaling-

up. Nonetheless, many chemical, electrochemical, and tech-

nological aspects have still to be elucidated and investigated.

In this paper, three exemplar MFCs have been chosen to

elucidate the phenomenological connection between pH

variations and power generated. A direct relationship with pH

is evident, with neutral pH being the best choice for the

startup of the MFCs. A pre-fermentation of the wastewater

can in principle speed the power production, by making the

environment completely anaerobic. Experimental evidence

thus demonstrated that the MFC, even if equipped with an

open-air cathode, is populated by anaerobic bacteria that use

sulfur in their lifecycle. SEM and EDX analyses demonstrated

the presence of sulfur in the cathodic electrode, and the

precipitation of carbonates on the surface. Despite a probable

clogging of the pores of the electrode by carbonates, a direct

chemical connection between the CO2 produced at the anode

and the not-increasing pH of the MFCs has been made.

Thermodynamic calculations have been made to understand

the accessible reactions and their connections with the actual

pH of the MFCs. The intimate connections between the

separate electrochemical responses of anodes or cathodes

and the possibility of getting under anodic or cathodic kinetic

control were demonstrated, showing an active involvement of

the pH in this process. Last but not least, a high coulombic

efficiency was obtained, demonstrating the effective involve-

ment of bacteria species (SRB included) in the power

generation.

Acknowledgments

This work has been financed by the Research Fund for the

Italian Electrical System under the Contract Agreement

between RSE and the Ministry of Economic Development e

General Directorate for Nuclear Energy, Renewable Energy and

Energy Efficiency. July 29, 2009 e Decree of March 19, 2009.

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