Chapter 9

Role of Biocathodes in Bioelectrochemical

Systems

V. Prakasam, S.G.F. Bagh, S. Ray, B. Fifield, L.A. Porter, and J.A. Lalman

9.1 Introduction

Environmental damage, depleting fossil fuels and energy security are major factors
driving intensive research efforts to develop carbon neutral or carbon negative
technologies which can be used to produce electricity and chemicals. Technologies
under development to achieve this goal include those based on bioelectrochemical,
biological, thermal and chemical processes. Evolving technologies employing
biological as well as electrochemical principles are grouped in the
bioelectrochemical systems (BESs) category. The main focus of this chapter is on
biocathodes used in BESs.
A BES such as a microbial fuel cell (MFC) is a two-chamber system consisting
of anode and cathode chambers separated by a proton exchange membrane (PEM).
This system is configured with an anode chamber containing electrochemically-
active microorganisms, while the cathode is abiotic. Microorganisms attached to
the anode oxidize electron donors such as glucose to produce electrons which travel
to the cathode. Protons are transported through the solution or across a membrane
separator. Depending on the application, BESs are configured with abiotic and
biotic cathodes (biocathode).

V. Prakasam • S.G.F. Bagh • J.A. Lalman (*)

Department of Civil and Environmental Engineering, University of Windsor, Windsor, ON,
Canada, N9B 3P4
e-mail: lalman@uwindsor.ca
S. Ray
Department of Chemical Engineering, National Institute of Technology, Agartala,
Jirania, Tripura, India, 79904
B. Fifield • L.A. Porter
Department of Biological Sciences, University of Windsor, Windsor, ON, Canada, N9B 3P4

© Capital Publishing Company, New Delhi, India 2018 165

D. Das (ed.), Microbial Fuel Cell,
https://doi.org/10.1007/978-3-319-66793-5_9
166 V. Prakasam et al.

Biocathodes can operate under aerobic, anoxic and anaerobic conditions. The
standard reduction potential for O2 to H2O is 0.818 V (Thauer et al. 1977). The O2/
H2O couple is more positive than the standard reduction potentials for sulphate/
bisulphide ( 0.217 V), nitrate/ammonia (0.360 V) and nitrate/nitrite (0.430 V). In
comparison, only nitrate/nitrogen reduction couple (0.760 V) is close to the O2/H2O
couple. Electrons at the cathode reduce O2 to H2O or H2O2 in MFCs, or water to H2
in microbial electrochemical cells (MECs).
Although the anodic oxidation reactions are similar, the different cathode reac-
tions result in MFCs producing electrical energy while in MECs, additional energy
is required to drive the overall reaction (Sasaki et al. 2011; Tartakovsky et al. 2009).
MFC technology can be classified into a wide array of technologies designated as
MXCs, where x is desalination, electrolysis and solar. Microbial desalination
cells (MDCs) are used in desalinating brackish water (Kim and Logan 2013)
while MECs are used for production of hydrogen (Tartakovsky et al. 2009) and
microbial solar cells (MSCs) are used for carbon dioxide sequestration (Pisciotta
et al. 2012).

9.2 BES Technology Utilizing Biocathodes

Abiotic cathodes constructed from platinum (Jeremiasse et al. 2010; Rozendal et al.
2008) are commonly used in MECs. However, limiting factors due to cost and
reduced catalytic efficiency caused by sulphide poisoning sulphide have forced
researchers to seek alternative options. BESs configured with biocathodes catalyze
electron transfer from cathodes to electro-positive terminal electron acceptors such
as oxygen or nitrate (Clauwaert et al. 2007a, b). Clauwaert et al. (2007a, b)
successfully employed BESs designed with bioanodes and biocathodes utilizing
inexpensive materials such as carbon or graphite rather than expensive metals such
as palladium. Additionally, biocathodes have also been configured into photosyn-
thetic MSCs to reduce carbon dioxide (Cao et al. 2009).
MDCs configured with biocathodes have been reported in several studies (Meng
et al. 2014; Wen et al. 2012). According to Meng et al. (2014), a biocathode
constructed from a graphite brush embedded with graphite granules was developed
for synergistic desalination, electricity generation and biosolids stabilization in an
MDC. These researchers reported a maximum power output reaching 3.2 W m 3
and an open circuit voltage (OCV) of 1.12 V. Wen et al. (2012) employed a
biocathode configured MDC and reported a maximum voltage of approximately
610 mV.
9 Role of Biocathodes in Bioelectrochemical Systems 167

9.3 Electron Acceptors and Microorganisms

Microorganisms accepting electrons directly or indirectly from a cathode are

referred to as electrotrophs (Lovley 2011; Logan 2009). BESs are operable using
a variety of electron acceptors such as oxygen, nitrate, sulphate, iron, manganese,
arsenate, fumarate, or carbon dioxide (Virdis et al. 2010; Freguia et al. 2010;
Rabaey et al. 2008; Clauwaert et al. 2007a).
Aerobic biocathodes variable performance and wide range of bacteria involved
with electron transfer are major issues under investigation by many researchers.
Mixed-community aerobic biocathode biofilms are a suitable substitute for chem-
ical catalysts at the cathode because of cost, robustness and sustainability. A wide
range of bacterial species belonging to the Alphaproteobacteria (Du et al. 2014;
Wang et al. 2013a; Zhang et al. 2011a; Clauwaert et al. 2007b), Betaproteobacteria
(Du et al. 2014; Wang et al. 2013b; Sun et al. 2012; Zhang et al. 2011b, 2012a;
Chen et al. 2008; Rabaey et al. 2008), Gammproteobacteria (Wang et al. 2013b,
2015; Du et al. 2014; Strycharz-Glaven et al. 2013; Chung et al. 2011; Zhang et al.
2011a; Chen et al. 2010; Schamphelaire et al. 2010; Rabaey et al. 2008; Clauwaert
et al. 2007b; Reimers et al. 2006), Bacteroidetes (Wang et al. 2013a; Sun et al.
2012; Xia et al. 2012; Chen et al. 2008, 2010) and other less well-known microor-
ganisms (Rimboud et al. 2015; Blanchet et al. 2014; Du et al. 2014; Wang et al.
2013a) have been identified as dominant in mixed community aerobic biocathodes.
Carbajosa et al. (2010) reported acidophilic Acidithiobacillus ferrooxidans is
involved with oxygen reduction in biocathodes. Other microorganisms such as
Chlorella vulgaris have been utilized under aerobic conditions (Campo et al.
2013; Wu et al. 2013).
Under anoxic conditions, nitrate and sulphate have been employed as electron
acceptors (Nguyen et al. 2015; Clauwaert et al. 2009). Denitrification and sulphate
reduction in BESs have been reported in many studies. Cai et al. (2014) reported
sulphate-reducing bacteria and homoacetogens were detected in MEC biocathodes.
Autotrophic hydrogen and sulphate consumer Desulfovibrio and planktonic micro-
bial consortia was detected on a biocathode configured BES (Pozo et al. 2015).
Nguyen et al. (2015) and Clauwaert et al. (2009) reported denitrification using
microorganisms attached to a cathode. Nitrifying bacteria detected on anoxic
biocathodes include Nitrosomonas sp., Nitrospira sp. and Nitrobacter
sp. (Du et al. 2014). Chen et al. (2010) reported using nitrate as electron acceptor
and the major microbial population detected included Alphaproteobacteria,
Betaproteobacteria, Gammaproteo-bacteria and Flavobacteria.
Jeremiasse et al. (2010) reported operating an MEC with the anode and cathode
catalyzing reactions mediated by microorganisms. These authors described the
formation of calcium phosphate and production of hydrogen plus methane. In an
MEC configured with a biocathode, microorganisms utilize the electrode as an
electron source to catalyze combining electrons and protons to produce hydrogen.
In other systems, microorganisms were able to produce methane from CO2 plus an
electron donor (Marshall et al. 2012, 2013; Pisciotta et al. 2012). Hydrogen and
168 V. Prakasam et al.

formate have been detected in mixed microbial communities autotrophic

biocathode configured MES reactors (Marshall et al. 2012, 2013; Zaybak et al.
2013; Pisciotta et al. 2012). Zaybak et al. (2013) used a BES configured with a
biocathode containing mixed microbial autotrophic community to produce butanol,
ethanol, hydrogen, acetate, propionate and butyrate.
Sulphate-reducing bacteria (SRB) as well as homoacetogens have been identi-
fied in MEC biocathodes (Kim et al. 2015). Sulphate reduction has been reported
using biocathodes with hydrogen as the electron donor (Coma et al. 2013; Yu
et al. 2008).
Photo-biocathodes are a promising option for combining photosynthesis into
BES systems (Xiao et al. 2015; Xiao and He 2014). BES configured with photo-
biocathodes has the potential of attaining the concept of a carbon neutral system.
Photo-biocathodes are useful for chemical production, carbon fixation (El-Mekawy
et al. 2014) and biomass production. Adding photosynthetic organisms is a means
for electron acceptors cathode as well as dissolved oxygen for the oxygen reducing
reaction (Campo et al. 2013; Gajda et al. 2013; Berk and Canfield 1964). Using
phototrophic biocatalysts in the cathode half-cell reaction is expected to permit
meeting the oxygen level requirements for the oxygen reducing reaction (Berk and
Canfield 1964) as well as producing biomass which utilized as a fuel for the MFC
anode. McCormick et al. (2011) and Xiao et al. (2015) reported cyanobacteria
Leptolyngbya and the green alga Acutodesmus as dominant photoautotrophs in
cathode suspension and biofilms.

9.4 Biocathode Materials

Electrode material properties are critical elements for the efficient and economical
feasibility for operating MFCs. Suitable materials properties should consider high
conductivity, low corrodibility, high specific surface area, suitable for microbial
growth and low cost (Wei et al. 2011a). Various materials utilized include graphite
felt (Zhang et al. 2012b), graphite granules (Zhang et al. 2011a), polyaniline (Ren
et al. 2013; Li et al. 2012), graphite fibre brush (Li et al. 2012; Zhang et al. 2011a,
b), poly(aniline-co-o-aminophenol) (Li et al. 2012), graphite plate (Behera et al.
2010; You et al. 2009), poly(aniline-co-2, 4-diaminophenol) (Li et al. 2012), poly
(aniline-1,8-diaminonaphthalene (Li et al. 2012), carbon felt (Li et al. 2012;
Schamphelaire et al. 2010), carbon paper (Zhang et al. 2012b), carbon fibre brush
(Tursun et al. 2016), granular carbon (semi coke) (Wei et al. 2011b) granular
activated carbon (Tursun et al. 2016; Sun et al. 2012; Wei et al. 2011b), carbon
felt cubes (Wei et al. 2011b), carbon nano tubes (Zhang et al. 2013), graphene nano
sheets (Ren et al. 2013) and stainless steel mesh (Zhang et al. 2012b; Zhang et al.
2013).
Tursun et al. (2016) compared graphite granules, activated carbon granules and
activated carbon powder and concluded that activated carbon granules show
improved power generation, higher chemical oxygen demand removal and
9 Role of Biocathodes in Bioelectrochemical Systems 169

coulombic efficiency. Zhang et al. (2012b) evaluated current density, power density
and polarization and reported graphite felt was the most effective biocathode when
compared to carbon paper and stainless steel mesh. In other studies, Zhang et al.
(2011b) compared the performance of graphite brushes, graphite granules and
graphite brushes plus graphite granules and concluded the startup time was less
for the graphite brushes plus graphite granules cathode configuration when com-
pared to graphite brushes. They also reported a maximum power density achieved
with a higher coulombic efficiency for the graphite brushes plus graphite granules
cathode.

9.4.1 General Material Characteristics

Electrode characteristics such as surface roughness, surface area, porosity, conduc-

tivity, and hydrophobicity are major factors affecting biofilm formation and, hence,
the performance of biocathodes.

9.4.1.1 Biocompatibility and Surface Roughness

Surface roughness can significantly impact the heterogeneity nature of biofilms on

surfaces. The ability to form and sustain biofilms on biocathode surfaces directly
impacts the electron transfer process. The structural heterogeneity and microbial
characteristics can influence the biofilm activities as well as the substrate and
product mass transfer (Yang et al. 2000). According to Dumas et al. (2008), surface
roughness enhances biofilm formation on graphite rather than on stainless steel and
the current density was much larger for the stainless steel electrode. Pons et al.
(2011) reported the biofilm structure significantly affecting current production with
isolated cells and small local colonies providing higher current density than dense
colonies.

9.4.1.2 Surface Area and Porosity

High surface area electrodes are affiliated with increasing power production (Logan
2009). Increasing porosity increases the surface area available for biofilm growth
(Santoro et al. 2014). Higher porosity reduces the diffusional resistance to the mass
transfer of oxygen, in case of aerobic biocathodes (Tursun et al. 2016).

9.4.1.3 Conductivity

Highly conductive materials reduce the resistance to the flow of electrons and
increases electron transfer. Higher resistance leads to a loss of energy in the form
170 V. Prakasam et al.

of heat. Highly conductive materials increase the electron transfer to micro-

organisms (Jourdin et al. 2014).

9.4.1.4 Hydrophobicity

The hydrophobic nature of the material can affect the type of microorganisms
attached to the surface (Mieke et al. 2013). Many microorganisms are capable of
changing their cell surface characteristics (charge and hydrophobicity) depending
on the environmental conditions (Marshall et al. 1971; Busscher and Weerkamp
1987). Bacterial adhesion can be enhanced by changing cathode properties such as
hydrophobicity as well as changing the pH and conductivity of the catholyte
(Rijnaarts et al. 1995; Van Loosdrecht et al. 1989).

9.5 Biofilm Formation

Biofilms are structured microbial communities encased and glued in a matrix of

complex extracellular biopolymers. A micro-colony, a basic unit of the biofilm, is
enclosed in a dense biopolymer attached to a surface (Costerton et al. 1995).
Biopolymer composition and the microbial populations in biofilms are major
factors affecting the electron transfer efficiency. A gradual decrease in the power
generation was observed with increasing cathode biofilm thickness on graphite
plate and stainless steel mesh (Behera et al. 2010). Biofilm formation is affected
by numerous factors such as reactor operational conditions, inoculum source,
environmental conditions, substrate and surface characteristics.

9.5.1 Biofilm Architecture

Biofilms architecture is also not always a uniform distribution of cells constructed

layer upon layer. Homogenous distribution of microorganisms on an electrode is
reported in many studies (Huang et al. 2011; Nevin et al. 2010). The availability of
nutrients plays an important role in biofilm formation as microorganism position
themselves according to the presence of nutrient gradients (Jain et al. 2007;
Costerton et al. 1995). Biopolymers can also enhance biofilm formation. Other
factors, including physicochemical properties (e.g. pH, temperature, viscosity and
flow rate) and the presence of other microorganisms can also affect biofilm devel-
opment (Patil et al. 2011; Costerton et al. 1995). Convective flow and turbulence,
caused by the roughness of the surface, allows for circulation within the biofilm.
Establishing biofilm growth on bioanodes and biocathodes by acclimatization of
electroactive bacteria (EAB) is a mechanism to increase establishment of micro-
colonies on surfaces (Xu et al. 2016; Wang et al. 2009; Kim et al. 2005).
9 Role of Biocathodes in Bioelectrochemical Systems 171

Acclimatization is useful in increasing the electrode performance by enhancing

biofilm attachment and growth and/or allowing the biofilm to attain a steady-state
open circuit potential (OCP) (Renslow et al. 2011; Larrosa-Guerrero et al. 2010).

9.6 Electron Transfer

Many microorganisms exchange electrons with solid surfaces or mediators. Elec-

trons are transferred between microorganisms, between microorganisms and sur-
faces and surface to microorganism. Electron acceptors reduction is achieved either
by bacteria or direct reduction on a biocathode.

9.6.1 Aerobic and Anaerobic Bacterial Electron Transport

Chains

Electron transport is mediated by a series of electron carriers which cascade

electrons from an electron donor with a relatively lower redox potential to an
electron acceptor with higher redox potential. The carrier system, localized in
bacterial plasma membranes, consists of linked electron carriers such as proteins,
flavins, cytochromes, non-heme iron components, other smaller non-protein car-
riers as well as multiple cytochrome oxidases (Kracke et al. 2015).
A generalized electron transport chain is initiated by the oxidation of NADH
(+H+) proceeds from flavoprotein dehydrogenase. Electrons are transported into a
non-heme iron centre and subsequently to ubiquinone to produce ubiquinol. The
non-heme iron centre receive electrons from ubiquinone, in case of aerobes, then to
cytochrome b1 and subsequently channelled to multiple cytochrome oxidases such
as cytochromes a, c4, c5 or d (Jurtshuk 1996). Configuration of these subsequent
carriers depends on the oxygen tension (Kurosu and Begari 2010).
In anaerobes, electrons from ubiquinone are transferred to cytochrome b1, to c1
and to c type cytochromes. Electrons from b1 may transfer to non-heme iron centre
or from c type cytochrome to cytochrome d and to the electron acceptor (Jurtshuk
1996).
Geobacter sulfurreducens and Shewanella oneidensis have been used as model
organisms to examine direct and indirect electron transfer mechanisms between
electrode surfaces (Ross et al. 2011; Rosenbaum et al. 2011; Thrash and Coates
2008; Bond and Lovley 2003). Outer-membrane cytochromes have been identified
as enabling compounds promoting electron transfer (Breuer et al. 2015; Shi et al.
2007, 2009). However, studies have shown significant differences in the different
electron transport chains in microorganisms. For example, soluble electron carriers
detected in Shewanella sp. are not present in Geobacter sp. (Marsili et al. 2008;
Holmes et al. 2006).
172 V. Prakasam et al.

9.6.2 Electrode-Microbe Electron Transfer Mechanisms

Microbe-anode and cathode-microbe electron transfer are similar but electron flows
in the opposite direction (Semenec and Franks 2015; Strycharz et al. 2011).
Electrons transfer from electrode to bacteria can be categorized into the following
(Choi and Sang 2016):
1. Direct electron transfer (DET)
2. Indirect electron transfer (IDET)
Microorganisms utilize several electron transfer modes such as: direct— involve
physical contact or attachment with nanowires, and indirect—use small organic
secreted by cells or added exogenously molecules as redox mediators or primary
metabolites or other intermediates (Patil et al. 2012).

9.6.2.1 Direct Electron Transfer (DET)

Direct Electron Transfer Through Surface Proteins

Outer bound, inner bound and trans-membrane proteins associated with electron
transport chains are involved with a series of redox reactions. Electron transfer
systems are not the same in microorganisms; however, these systems essentially
perform the same function. Although electron transfer systems in Geobacter
sulfurreducens can function on both anodes and cathodes (Geelhoed and Stams
2011; Strycharz et al. 2011; Nevin et al. 2009), it is unclear if the same proteins are
involved.
Carriers mediating electron transfer reactions include an extracellular site on the
outer membrane designated as MtrC, MrtA, a periplasmic c-type cytochrome,
CymA, positioned on a site between the inner membrane and the periplasm and
OmcA, a protein located on the inner membrane (Choi and Sang 2016). OmcS and
OmcF, two of the most abundant proteins from the outer surfaces of intact c-type
cytochrome cells, are affiliated with the electron transfer process.
Direct electron transfer from cathode to hydrogenases via cytochromes has been
proposed by Rosenbaum et al. (2011). Genomic sequence of Desulfovibrio vulgaris
has shown to possess genes coded for various c-type cytochromes (Heidelberg et al.
2004). These cytochromes may create a network of heme centres in electron
transport chains. Dinh et al. (2004) observed a Desulfobacterium isolate and a
Methanobacterium which can accept electrons from metallic iron. Various types of
cytochromes have been detected in the outer membrane of Desulfovibrio sp. and
their role as redox partners of hydrogenase was confirmed by many researchers
(Barton et al. 2007; Guiral-Brugna et al. 2001; Pereira et al. 1996). These findings
form the basis for a mechanism of cytochromes accepting electrons from cathode
and coupling to hydrogenases producing hydrogen in electron transport chains.
9 Role of Biocathodes in Bioelectrochemical Systems 173

Direct Electron Transfer Through Conducting Pili

This direct electron transfer mechanism relies on conductive pili (nanowires) for
electron transfer. Nanowires may function by enhancing the formation of thicker
electroactive biofilms which assist with charge transfer. Nanowires have been
reported in Geobacter and Shewanella strains (El-Naggar et al. 2010). In brief,
DET occurs when active sites on the membrane/extracellular proteins are bound or
close to the electrode surface.

9.6.2.2 Indirect Electron Transfer (IDET)

In microbial systems, electron transfer is mediated through chemical addition

(artificially), by-products as well as extracellular polymeric substances (EPS)
(Park and Zeikus 1999; Park et al. 1999; Pequin et al. 1994; Kim and Zeikus
1992; White et al. 1987).
Mediator Shuttled Electron Transfer
Indirect electron transfer (IDET) is associated with the electron transport via shuttle
chemicals. These mediating chemicals adsorb and facilitate the transfer of electrons
from carbon and metal surfaces to microorganisms. Many redox dyes, such as
methyl viologen (MV) (Pequin et al. 1994; White et al. 1987), benzyl viologen
(White et al. 1987), and neutral red (Kim and Zeikus 1992) with redox potential
lower values than NAD can affect biological redox reactions in bacterial systems.
For example, methyl viologen, an artificial mediator, was shown to increase ethanol
production from acetate at a cathode (Steinbusch et al. 2010).
EPS Mediated Electron Transfer
Extracellular polymeric substances (EPS) have been shown to function as a means
for electron transport from cathode surfaces to micro-organisms. This property has
become basis for many studies in improving cathode material for maximum power
production. Cathode surfaces modified with oligosaccharides or polysaccharides
and other polymeric materials have been reported to increase the specific surface
area, alter the charge of cathode material and improve efficient electron transfer
(Ren et al. 2013; Li et al. 2012).

9.6.2.3 Proteins Affiliated with Extracellular Electron Transfer

In addition to membrane bound cytochromes, other proteins such as ferrodoxin

oxidoreductases, hydrogenases coupled with ferrodoxins, methyl transferases,
rubredoxin, fumarate reductase, and formate dehydrogenase participate in electron
transfer (Choi and Sang 2016).
174 V. Prakasam et al.

9.7 Microbial Characterization Methods

Genomic/metagenomic, transcriptomic/metatranscriptomic, proteomic/meta-

proteomic, metabolomics/meta-metabomoics, microscopic coupled with flow
cytometry are employed to understand microbial interactions as well as metabolism
in microorganisms. Combined metagenomic and metaproteomic analyses can be
particularly useful for generating information related to the functions of biofilm
constituents. Metaproteomics is useful because the data can provide functional
information from electrode-grown cells in order to understand biofilm electron
transfer pathways.

9.7.1 Biofilm Characterization

A variety of techniques, including biological, microscopic and chemical, are

available for characterizing and for community analysis of biofilm on electrode
surfaces. Each method has different level of taxonomical resolution and, therefore,
a specific application purpose. Each technique is classified into the following three
levels of detection: microbial detection, assessing and characterizing the commu-
nity composition and characterization based on functional genes and microbial
activity.

9.7.2 Microorganism Detection

Microbial communities can be analyzed using both culture-dependent and inde-

pendent approaches. Isolation of specific populations is accomplished using a
variety of methods. Cultures are serially diluted and plated on solid medium
where single colonies are subsequently cultured further as a pure culture. Further,
culture independent techniques are needed for complete detection of populations of
microbes.
Another powerful tool used to detect and quantify microbes in a biofilm makes
use of fluorescent markers is fluorescence in situ hybridization (FISH) (Moter and
G€obel 2000). In this technique, fluorescent oligonucleotide probes are designed
against specific known sequences such as genus specific sequences. Many varia-
tions of FISH have been developed to enhance the signal generated from the
sample, which is especially useful if a low microbial population is predicted
(Pernthaler et al. 2002). Quantitative real time polymerase chain reaction
(q-PCR) is used to rapidly determine and detect specific microbes within a biofilm
(White et al. 2009).
9 Role of Biocathodes in Bioelectrochemical Systems 175

9.7.3 Composition and Characterization of Microbial

Communities

Detection and determination of the relative abundance of microbes within a com-

munity is not sufficient for complete characterization of the population. The gold
standard in identifying cultures is 16S rRNA sequencing (Rosselló-Mora and
Amann 2001; Amann et al. 1995; Woese et al. 1985). The highly conserved regions
of 16S rRNA allows for reliable universal primers, while variable regions can be
used for comparative taxonomy (Prakash et al. 2007). Identification of the isolates
is examined by comparing the 16S rRNA gene sequence to international public
sequence databases (e.g. GenBank). Genetic fingerprinting techniques are com-
monly employed to characterize the composition of a microbial community. Gel
electrophoresis techniques are used to separate the amplicons based on their
sequence. Denaturing gradient gel electrophoresis (DGGE) relies on an increasing
gradient of denaturants (formamide and urea) to separate amplicons; alternatively
non-denaturing electrophoresis is used to separate these fragments while
maintaining their secondary structure. Temperature gradient gel electrophoresis
(TGGE) uses a temperature gradient for amplicon separation.
An alternative genetic approach includes terminal restriction fragment length
polymorphism (T-RFLP). T-RFLP is based on the amplification of target genes,
typically the hyper-variable region of 16S rRNA genes from the bulk population
(Liu et al. 1997). Another version of RFLP is amplified ribosomal DNA restriction
analysis (ARDRA). This technique is another extension of RFLP where conserved
regions at the end of the 16S gene are amplified and digested with restriction
enzymes (Vaneechoutte et al. 1992; Vaneechoutte et al. 1993).
Single-strand conformation polymorphism (SSCP) analysis is a simple and
sensitive technique for examining genetic variations within microbial communities
(Widjojoatmodjo et al. 1995; Orita et al. 1989). The SSCP method is based on
analyzing an altered conformation in comparison to a defined conformation. A
single base substitution leads to altered mobility of the single-stranded DNA under
non-denaturing electrophoresis. The differential intra-molecular folding results in
the banding pattern obtained from single-strand conformation analysis.
Genomic methods dedicated towards extraction of nucleic acid and its subse-
quent analysis have been used to characterize microbial community. Metagenomics
is used to analyze the collective genome from microorganisms in an ecosystem.
An indirect method to delineate differences between bacterial species is using
the percent similarity via DNA-DNA hybridization. With increasing number of
sequenced microbial genomes the direct determination of the similarity between
entire genome sequences may replace DNA-DNA hybridization (Goris et al. 2007;
Vandamme et al. 1996).
Genetic approaches can yield a great deal of information regarding the charac-
terization of biofilms. However, further classification based on phenotypic or
176 V. Prakasam et al.

chemotaxonomic characterization is required. An example of chemotaxonomy is

cellular fatty acid methyl ester (FAME) analysis. FAME analysis is performed
using gas chromatography (GC) or liquid chromatography (LC) coupled with mass
spectrometry (MS) (Vandamme et al. 1996).

9.7.4 Analysis of Functional Genes and Activity of Microbes

When assessing and characterizing a biofilm, not only is the structure and compo-
sition important, the functions these microbes perform and their activity are also
crucial to fully understand these populations. To achieve this, techniques based on
metatranscriptome and metaproteome are used. Metatranscriptomics can be used to
quantify levels of gene expression within complex microbial communities in a high
throughput manner. Combining metaproteomic and metagenomic analyses is con-
sidered useful for gathering information of constituents and functions of constitu-
ents in a biofilm (Wang et al. 2015).
For metatranscriptomic techniques, RNA is isolated from the population and
analyzed via real time PCR (RT-PCR) or microarray analysis (Sharkey et al. 2004).
Using RT-PCR, changes in gene expression of functional genes in response to
varying environmental conditions can be assessed (Sharkey et al. 2004). The
microarray based metatranscriptomic analysis allows for a large number of target
sequences to be analyzed at one time, provide a rapid, high throughput method of
analyzing expression of functional genes.
Metaproteomic data is able to produce similar information as
metatranscriptomics. However, metaproteomics can also provide information at
the level of protein expression (Wilmes and Bond 2006). Specific functions and
activities performed by microbes are known to be associated with expression of
particular sets of proteins; thus functional activity of the microbes can be analyzed.
Stable isotope probing (SIP) can be used to identify the active populations within
a microbial community (Dumont and Murrell 2005; Radajewski et al. 2000). Stable
isotopes (13C or 15N) which have successfully metabolized and incorporated the
substrate can be separated by density gradient centrifugation. Isolated populations
can then be analyzed using genetic approaches to determine the strain of microbe
response for metabolizing the substrate.

9.7.5 Polyphasic Taxonomical Approach

Using the polyphasic approach to classify microbes undoubtedly enhances the

accuracy of establishing phylogenetic relationships between microbes, as well as
in characterizing new species. This approach uses many of the techniques described
9 Role of Biocathodes in Bioelectrochemical Systems 177

in the above sections including complete gene sequencing and comparative analysis
by phylogenetic trees, DNA-DNA hybridization, analyses of molecular markers,
biochemical assays and microscopic characterization to gather collective informa-
tion on microorganisms (Prakash et al. 2007; Vandamme et al. 1996). Polyphasic
taxonomy is strengthened by further integrating genotypic, phenotypic and phylo-
genetic microbial data in a stepwise manner.

9.7.6 Microscopic Methods

Light microscopy techniques have dramatically improved visibility and contrast in

unstained and living material. Adaptions to microscope hardware have made
contrast imaging possible; one example is the Hoffman Modulation Contrast
(HMC) which creates a 3D image by placing an amplitude filter in the objective
of the microscope. The result is a high resolution three-dimensional image capable
of optical sectioning, directionality and control over contrast and coherence (Hoff-
man 1988).
Scanning electron microscopy (SEM) is a powerful technique capable of exam-
ining the surface structure of biological samples and often used for visualization of
microorganisms in a biofilm. Environmental SEM (ESEM) is preferred for exam-
ining biofilms because of issues related to the water of hydration with the vacuum
system. Energy x-ray spectroscopy (EDS) is another useful analytical tool inte-
grated with SEM.
Transmission electron microscopy (TEM) allows quantitative analysis at high
resolution across the sections of the biofilms and, thereby, deriving valuable
information on the spatial arrangement and cellular ultra-structure.
AFM is a type of near-field scanning probe microscope which uses a sharp probe
or tip to map the contours of a sample and is therefore not limited in resolution due
to diffraction effects. The AFM technique is very useful since it allows the precise
measurement of dimensions of individual bacteria (Surman et al. 1996; Nivens et al.
1995).
Confocal scanning laser microscopy (CSLM) has improved the analytical
precision of light microscopy. CSLM is well suited for studying microbial biofilms
in that the in-situ, nondestructive analysis of living, fully hydrated biofilms is
performed without chemical fixation or embedding. Combining confocal micro-
scope capabilities with fluorescence in situ hybridization (FISH), which uses
fluorescently labelled oligonucleotide probes to identify specific microbial com-
munity members, allows for accurate and quantitative analysis of multi-species
biofilms in both natural and artificial environments (Almeida et al. 2011; Yang
et al. 2011).
178 V. Prakasam et al.

9.7.7 Spectroscopic Methods

Fourier transform infrared spectroscopy (FTIR) is used to study the interaction

between IR radiation and the vibrational modes of molecules in condensed phases
(solids and liquids). Commercially available IR instruments are capable of studying
aqueous biofilms. Coupling of attenuated total reflection (ATR) sampling technique
with FT-IR spectrometry allows examination of biofilms in aqueous environments;
however, the water background signal requires subtracting. Surface-enhanced
Raman scattering (SERS) is another technique which is used to gather information
on microorganisms (Ivleva et al. 2008).

9.7.8 Nuclear Magnetic Resonance Imaging

Nuclear magnetic resonance imaging (NMRI) works on the principle of tracing the
water movement within the biofilm. Intra-biofilm flow is known to profoundly
affect mass transport within biofilms. NMRI is useful in understanding molecular
diffusion and biofilm process modelling (Costerton et al. 1995).

9.7.9 Flow Cytometry

Flow cytometry as a method for microbial community characterization is beginning

to gain more attention in recent years. In brief, samples are stained with fluorescent
dyes and then examined using a series of lasers. Detectors are able to determine
intensity of the fluorescent signal, which is subsequently used to quantify either the
relative abundance of microbes in a particular sample (Hammes et al. 2008), or can
also be used to determine the composition of samples (Kerstens et al. 2015;
Ksontini et al. 2013).
To enable identification of specific microbial species within a community,
combining flow cytometry with FISH, known as flow-FISH, can be utilized
(Nettmann et al. 2013; Amann et al. 1990). In this technique, fluorescently labelled
probes with sequences specific to 16S rRNA sequences are used to label a microbial
community. Samples are then analyzed using a flow cytometer where the expres-
sion and quantification of the fluorescent probe can be readily and rapidly evalu-
ated, allowing for quick identification of microbial species in a given community.
9 Role of Biocathodes in Bioelectrochemical Systems 179

9.8 Conclusions

BESs are an evolving technology which remains in its developmental stages. A

crucial factor affecting developing and implementing the technology is the cathode.
Abiotic cathodes and biocathodes have been evaluated in BESs. Numerous advan-
tages have been attributed to using biocathodes when compared to abiotic cathodes.
Biocathodes are a crucial design element affecting efficient electron transfer and,
hence, electricity production. Advancing the technology requires a thorough under-
standing of how material characteristics as well as microbial factors affect electron
transfer. Investigating electron transfer mechanisms has been evaluated using
numerous biological and chemical tools.

Acknowledgements Dr. Srimanta Ray was a visiting research associate in the Department of
Civil and Environmental Engineering, University of Windsor, Ontario, Canada during preparation
of this manuscript. Financial contribution for Dr. Ray’s associateship was provided by the
Department of Biotechnology, Government of India. The Canadian Queen Elizabeth II Diamond
Jubilee Scholarship program provided funding to support Mr. Vignesh Prakasam. Funding to
support Ms. Fatemeh Bagh was provided by the Ontario Trillium foundation.

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