i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3

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Review

Biocatalysts in microbial electrolysis cells: A review

Masoud Hasany, Mohammad Mahdi Mardanpour, Soheila Yaghmaei*

Department of Chemical and Petroleum Engineering, Sharif University of Technology, Azadi Avenue, P.O. Box 11365-
9465, Tehran, Iran

article info abstract

Article history: Microbial electrolysis cells (MECs) are bioelectrochemical reactors in which chemical en-
Received 28 August 2015 ergy stored in organic compounds are converted to hydrogen through biocatalytic oxida-
Received in revised form tion by microorganisms. The performance of MECs is highly affected by microbial
25 October 2015 communities that are impartible parts of this technology. A better understanding of mi-
Accepted 25 October 2015 crobial interactions and competitions mechanisms, has aided the comprehension of ideas
Available online 14 November 2015 and guidelines for cost effective commercial scales design. In this study, a comprehensive
review of current knowledge in the microbial characterization, enrichment, and evaluation
Keywords: of effective parameters of microbial community in microbial electrolysis cells for typical
Microbial electrolysis cells biohydrogen production is summarized. Some existing challenges of biocatalysts behaviors
Microbial community in MECs (such as undesirable microorganisms growth and deactivation of desirable spe-
Hydrogen cies) were disclosed, and outlooks for future research were also proposed, which would aid
Methanogens the acquisition of knowledge and contribute to the development of MECs and their
application in bioenergy production with simultaneous treatment of wastewater.
Copyright 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1478
2. Microbial community analysis methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1478
3. Inoculation methods, pure and mixed cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1480
4. Effective parameters on microbial communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1482
4.1. Effect of anode and applied potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1482
4.2. Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483
4.3. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484
4.4. Physical and chemical characteristics of electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484
4.5. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484
5. Methanogens and their controlling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1485
5.1. The ways to reduce methanogens activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1485
6. Biocathodes in MECs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1488

* Corresponding author. Tel./fax: 98 2166166430.

E-mail address: yaghmaei@sharif.edu (S. Yaghmaei).
http://dx.doi.org/10.1016/j.ijhydene.2015.10.097
0360-3199/Copyright 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1478 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
7. The MFC-MEC coupled systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1489
8. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1489
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1490
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1490
Introduction
In the last decade, academic researches about bio-
electrochemical treatment of various wastewaters with
simultaneous bioenergy production have increased signifi-
cantly. Nowadays, most academic studies are assigned to
microbial fuel cell (MFC) and microbial electrolysis cell (MEC)
technologies for production of electricity and hydrogen
respectively and wastewater treatment simultaneously [1].
Microbial fuel cells (MFCs) and microbial electrolysis cells
(MECs) are bioelectrochemical reactors in which chemical en-
ergy stored in organic compounds are converted to electricity
or hydrogen through catalytic oxidation by microorganisms.
Therefore, the use of this technology in wastewater treatment
in addition to organic contaminants removal should lead to
clean energy production; proof of this has motivated great in-
terest in this field in recent years [2e4]. Microorganisms as
biocatalysts extract electrons and protons by oxidation of
organic content and produce CO2 as a by-product. Electrons are
transferred to the anode surface and then conducted to the
cathode through an external circuit. In MFCs, protons are also
transferred to the cathode by a proton exchange membrane
(PEM), and are then reduced to water molecules in the presence
of oxygen. If the cathodic chamber becomes anaerobic, the cell
will change to a MEC and the transferred protons will be
reduced to hydrogen as the end product. The final reduction of
protons to hydrogen is a thermodynamically non-spontaneous
reaction; hence it requires an external energy input.
Higher energy efficiency of these technologies compared to
fermentation systems and acclimatization to treatment of a
wide range of wastewaters has enhanced academic interests
[5e7]. Therefore, in some researches, these technologies were
used as a complementary system to other processes [7e10].
Nevertheless, there are a lot of challenges in commercializa-
tion of these novel technologies for bioenergy production and/
or wastewater treatment [11,12].
As regard the commercialization of MECs and MFCs,
comprehensive studies on microbial communities as bio-
catalysts of bioelectrochemical processes, that are impartible
parts of these technologies, is absolutely helpful. As a matter
of fact, investigation on microbial community has been done
with the aim of better understanding of chemical and
biochemical interactions in the systems (such as substrate
degradation by biocatalysts and competition between various
microorganisms) and also figuring out the ideas and guide-
lines for designing of commercial scales.
In recent years, the numbers of academic publications on
the investigation of microbial community in MECs, and its
effects on energy recovery have increased dramatically. It
should be noted that the most important investigation on
microbial communities in MECs and MFCs is about
characterization of cathodic or anodic microorganisms which
have significant roles in bioenergy production.
The investigation of microbial community in MEC re-
searches is mainly less in comparison to MFC. Most of these
studies pointed out that there is no difference between mi-
crobial communities of MECs and MFCs and that maybe as a
result of the same conditions of anodic chamber in both sys-
tems. On the other hand, the main difference between MFCs
and MECs, is anaerobic condition of cathodic chamber in MECs
and implementation of external potential to the system, may
have significant impact on cathodic and even anodic microbial
communities in MECs. As a consequence, the competition
between methanogenic microorganisms (including aceto-
clastic and hydrogenotrophic microorganisms) as well as
exoelectrogens ones can seriously impact on MECs operation.
Understanding how the exoelectrogen bacteria commu-
nities develops and changes over time, in terms of coloniza-
tion, invasion and succession, will give birth to the totally new
horizon of insight the bioelectrochemical processes in regards
to the developed novel applications and commercialization of
MECs.
Despite several review studies in MFC that concern bio-
catalysts [13,14], extracellular electron transfer mechanisms
[15,16], substrates [17], electrodes [18], configurations [4,19,20]
and future prospects [3,21e23], no review study found that
focuses on MECs specially their biocatalysts. In view point of
the main differences between MEC and MFC (such as microbial
communities and the half cathodic reaction), present study is a
first comprehensive review of current knowledge in the mi-
crobial characterization, enrichment, evaluation of effective
parameters on microbial community, the symbiosis of
different microbial groups and ways to dominate electrogen-
esis group in microbial electrolysis cells for typical bio-
hydrogen production. Some existing challenges of biocatalysts
behaviors in MECs (such as undesirable microorganisms'
growth and deactivation of desirable species) were disclosed,
and outlooks for future research were also proposed, which
would aid the acquisition of knowledge and contribute to the
development of MECs and their application in bioenergy pro-
duction with simultaneous treatment of wastewater.
Moreover, for the first time, methanogenesis as the most
important issue in performance of MECs was extensively
investigated by summarization of all the studies in this field
and the used methods for addressing this problem.
Microbial community analysis methods
Until now, various methods have been used for identification
and analysis of microbial communities in MECs. These
methods are summarized in Table 1. Moreover, more simple
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3 1479

Table 1 e Used methods in microbial community analysis of MECs.

Method Characteristics References
16S ribosomal RNA oligonucleotides cataloging Existence or inexistence of microbial genes [26,31e36]
16S ribosomal DNA oligonucleotides cataloging Existence or inexistence of microbial genes [25,37]
Denaturing Gradient Gel Electrophoresis (DGGE) Qualitative and semi quantitative comparison of bacterial species [30,38]
Terminal Restriction Fragment Length Reproducible, qualitative and semi quantitative comparison of bacterial [34]
Polymorphism (T-RFLP) species
Automated Ribosomal Intragenic Spacer Reproducible, qualitative and semi quantitative comparison of bacterial [34]
Analysis (ARISA) species
Formyl-Tetrahydrofolate Synthetase (FTHFS) Identification of acetogenesis bacteria [39,40]
Fluorescence In Situ Hybridization (FISH) Visualization and mapping the genetic material in an individual's [35]
bacterial cells
Single-Strand Conformation Polymorphism (SSCP) Reproducible, qualitative and quantitative identity of bacterial species [28,34,41,42]
GS-FLX yrosequencing of the 16S rRNA gene 454 Reproducible, qualitative and quantitative identity of bacterial species [43]
Quantitative Real-Time PCR (QPCR) Quantitative gene expression analysis, genotyping and detection of [44e47]
bacteria
Quartz Crystal Microbalance (QCM) Measuring a mass variation per unit area of biofilm [48]
GeoChip Structural, metabolic potential, and diversity analysis of microbial [41]
communities

analysis such as comparison of microbial biomass enrich-
ment, porosity, color, arraignment and structure of biofilm,
and analysis of other matter around the microorganisms (like
EPS (Extracellular polymeric substance)) by using scanning
electron microscopy (SEM) [24e28], transmission electron
microscopy (TEM) [29] and single-strand conformation poly-
morphism (SSCP) [30] have been used.
For the first time, Croese et al. [49] reported that the use of
redundancy analysis (RDA) as a multivariate statistics method
for analysis of bioelectrochemical systems and comparison of
the obtained results with DGGE could help improve knowl-
edge of microorganisms and its interaction with electrodes in
MECs. It is true that the prominent bands in DGGE results
represent prominent species in different areas of the anode
electrode, but the most prominent band is not necessarily the
most important in MEC efficiency. Thus, RDA was used to
determine the microbial species with more important roles in
MEC efficiency [49]. In their research, isolates of five different
MECs with different anolyte flows (perpendicular and parallel
to anode electrode) and also the effects of using different
membrane (including anion exchange membrane (AEM) and
cation exchange membrane (CEM)) on microbial community
were analyzed with DGGE. The results showed high variety of
microorganism and big differences between sampled micro-
organisms from anolyte and biofilm. The community
composition which was most strongly correlated with current
density and Clostridium sticklandii strain, and has been detec-
ted in other studies [50,51] had a major role in current pro-
duction from acetate at the anodic compartment. There was
the same archeal community in different MECs. Also, FISH
and SEM pictures showed that there has been rod shape
Eubacteria and clusters of archaea (similar to Methanosarcina)
on electrode surface and in the space between strings of
graphite felt (anode electrode), respectively [49]. Based on this
observation, Croese et al., pointed out that there is a proba-
bility that archaea had a negative role in electron production.
However, there are research works in which electricity pro-
duction by suspended microorganisms had been reported [42].
In addition, current production by archaea in MFC had been
confirmed by Abrevaya et al. [52].
The electrochemical quartz crystal microbalance (QCM)
can be used for monitoring the development of a microbial
electrolysis cell bioanode. With this method, microbial com-
munity development and current generation by electrogenic
microorganisms can be investigated simultaneously. In this
way, Kleijn et al. [48] showed that anodic biomass and current
density had increased simultaneously. The total frequency
shift in the growth phase was 785 Hz, corresponding to an
increase in adhering biomass of 13.9 mg/cm2. While the total
frequency shift from the start of the experiment was 920 Hz,
corresponding to a biomass of 16.3 mg/cm2. For development of
biofilm, cells density and average thickness were about 1 g/
cm3 and 0.16 mm, respectively [48].
The use of GS-FLX pyrosequencing of the 16S rRNA gene
gives a more extensive vision of microbial communities'
structure in bioelectrochemical systems [40,45]. In some aca-
demic works, rare species were observed such that charac-
terization of these species was not achieved. That is as a result
of the limitations of conventional analysis methods (such as
DGGE) [53]. Nevertheless, the possibility of extensive cooper-
ation of these uncharacterized microorganisms in energy
production is high. Lu et al. [45] used waste activated sludge
(WAS) as a substrate in MEC. They investigated the microbial
communities of raw WAS, anode biofilms in the WAS-fed
MECs and anode biofilms in the alkaline-WAS-fed MECs
using 454 GS-FLX pyrosequencing of the 16S rRNA gene. Also,
a quantitative analysis of bacteria, archaea and methanogens
were performed with QPCR. The results showed that the
dominant populations in MECs were more diverse than those
that were inoculated and fed with WAS and there was a clear
distinction between MECs and WAS as an inoculation media
in the microbial community structure. Diverse acid producing
bacteria and exoelectrogens were detected in the MECs while
they were rarely found in WAS. The symbiosis of these groups
in MEC biofilm led to degradation of a wider range of organic
matter (such as carbohydrates and proteins) in WAS and
production of more hydrogen compared to fermentation sys-
tems. It should be noted that the configuration of MECs
(including structure, electrodes, membrane, and etc) was
significantly impacted in microbial communities. Lu et al., did
1480 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
not identify many common species in the three types of mi-
crobial communities from MECs and WAS, but Proteobacteria
and Bacteroidetes were the most common. Generally, each of
the three microbial communities had great diversity of spe-
cies, so 25 phyla were identified. Most of the differences be-
tween the three types of microbial communities were
differences in terms of the distribution of phyla Bacteroidetes,
Firmicutes, and Proteobacteria in the total community compo-
sition. Despite the fact that exoelectrogens like Geobacter sp.
usually belong to Proteobacteria, however, Proteobacteria was
much more in raw WAS in comparison to MECs biofilms.
Nitrospirae and Actinobacteria were more predominant in raw
WAS and MECs biofilms, respectively. High level of acetate
concentration in alkaline-WAS-fed MEC led to predominance
of acetoclastic methanogens. However, hydrogenotrophic
methanogens whether in raw-WAS-fed MECs or alkaline-
WAS-fed MECs were numerous. Also, two hydrogenotrophic
methanogens orders including Methanobacteriales (MBT) and
Methanomicrobiales (MMB), and two acetoclastic metha-
nogens families including Methanosarcinaceae (MSC) and
Methanosaetaceae (MST) were all detected in the three types
of microbial communities.
Inoculation methods, pure and mixed cultures
Inoculation of MECs and MFCs with mixed culture especially
when wastewaters are used as substrate is more general
compared to pure culture inoculation. This is because a vast
range of organic materials can be degraded by mixed cultures
which provide higher energy efficiency and better removal
method of organic pollutant from wastewaters. Besides these
advantages, existence of some microorganisms such as
methanogens and nonexoelectrogens in mixed cultures can
effectively reduce energy efficiency in some cases.
The work of Liu et al. can be presupposed as the first study
in which MECs microbial community were investigated [28].
The low hydrogen recovery efficiency of the MEC (11.6%)
attributed to the presence of suspended growing microor-
ganisms and competition among other cations through the
proton exchange membrane. In this case the produced elec-
trons and protons did not find the opportunity to pass the
anolyte and be reduced to hydrogen [28]. In contrast to these
observations, Wang et al. reported the presence of these mi-
croorganisms as an advantage to hydrogen production [42].
They emphasized that decreasing the suspended microbial
flow to anolyte compartment, terminate hydrogen production
and enhance biofilm detachment. Therefore, more experi-
mental works is required for better decisions.
Using pure cultures for inoculation is preferred especially
when the aim is for investigation of molecular mechanisms of
microorganisms in electron transportation [54e58]. In addi-
tion, while removing a selected material without degradation
of other compounds, selection of the pure and suitable
microorganism species is very important [10].
Since the main goal of most MECs studies is wastewater
treatment, using of mixed culture is more conspicuous
[28,59,60]. Generally, most wastewater contains high amounts
of microorganisms which are very suitable for its biological
treatment. That is why inoculum used in most MECs and
MFCs works is taken from wastewater treatment plant
[28,35,42,61,62].
In many works, the used anode electrode in an MFC which
took part as an electron acceptor, has been transferred to a
new MEC [31,33,43,62,63]. This act led to acclimation of suit-
able microorganisms as biocatalysts and exploitation of a well
formed biofilm [59]. Fu et al. showed that transfer of a used
biocathode to other systems is beneficial provided that the
biocathode had been employed in a cathode role in the pre-
vious system. Otherwise, desirable performance will not be
attained [64]. However, there are some cases in which the
utilized anode has been used in a new MEC as cathode [1,63].
For this purpose, the suitable substrates in a successive pro-
cedure should be used to adjust desirable microorganisms as
biocatalyst in the biofilm; in the next step the poles of the
system must be reversed. In this way, the microorganisms in
new biocathode can catalyze the biohydrogen evolution pro-
cess [1,63]. In some studies, however, the utilized MFC,
without any major alternation, was used for MEC applications
[65]. Also, one can use the anaerobic chamber outlet of a MEC
or MFC for inoculation of new systems [38,66,67]. These
methods lead to faster formation of anodic or cathodic bio-
films and the onset of the bioenergy production in a shorter
time [41].
Comparison of biocatalytical performance of two pure
cultures (Geobacter sulfurreducens and Geobacter metallireducens)
and one mixed culture based on microbial communities and
bioenergy parameters (such as coulombic efficiency, bio-
hydrogen recovery, bioenergy recovery and biohydrogen pro-
duction) investigations were reported by Call et al. [31]. The
results showed that MECs inoculated with mixed culture and
G. sulfurreducens produced the same amount of hydrogen gas.
But the one inoculated with G. metallireducens produced lower
hydrogen. Also, the MEC containing mixed culture before the
beginning of methane production, had the most coulombic
efficiency and bioenergy recovery. The 16S rRNA microbial
analysis showed that despite the low count of G. sulfurreducens
in mixed culture inoculum, after some time, 72% of the MEC
biofilm colonies allocated to G. sulfurreducens. The SEM mi-
crographs showed different biofilm morphologies of the
mixed culture and G. sulfurreducens. The mixed culture formed
compact and tight biofilm on the surface of graphite fibers
than that of G. sulfurreducens. Thus, mixed culture achieved a
better contact with the electrode surface. They also showed
inoculation with pure culture unlike the mixed culture with
more anodic potential, current density and biohydrogen gas
could be obtained in lower applied voltages [31].
Using of mixed culture increases the probability of having
the presence of unknown species besides the known species
such as Geobacter and Shewanella. However, in some studies
the presence of species similar to Shewanella sp. has been
confirmed and this was related to microbial tendency to
consume lactate and low concentration of lactate as a medi-
ator substrate in an anaerobic medium [68]. In the beginning,
it was thought that the species on anodic biofilm were only
iron-reducing bacterium (i.e. Geobacter [69,70] and Shewanella
[57,71e73]); but, further results revealed that there were some
microorganisms which affect electron production and trans-
portation. Interestingly, some species similar to Geobacter
have a complementing role in biofilm and are necessary for
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
energy generation [35,60,74]. These genera have outer mem-
brane cytochrome and conductive pili to aid their comple-
mentary role. In addition to these species, Pseudomonas [75],
due to its ability of produce of self-produced mediators which
accelerate the electron transport, are worthy of being utilized
in the bioelectrochemical systems [41]. Generally, the bio-
electrochemical processes depend on special features of
various microorganisms as biocatalysts and it does not
depend on a specific microorganism and its special features or
its surrounding conditions [33]. The symbiosis of the other
microorganisms with iron-reducing bacteria can be attributed
to the fact that a few substrates can be utilized by iron-
reducing bacteria to biodegrade and produce electrons.
Thus, the existence of other microorganisms plays comple-
mentary role in expanding the range of utilizable organic
materials for biodegradation and improving bio-
electrochemical cells efficiency. Even some studies have
revealed that the produced metabolites by some species can
be used by other species in energy production. For instance,
the Phenazine molecules produced by Pseudomonas can be
used by other microorganism as electrons shuttles for their
transportation to anode electrode [74,76].
In addition, it was demonstrated that the combination of
mixed cultures improves MEC efficiency. Miceli et al. [77],
combined a butyrate oxidizing community with a Geobacter
rich culture to generate a biocatalyst community which had
been reported in literature for high current production from
butyrate and able to reach a current density of 11 A/m2 and a
coulombic efficiency of 70%. Microbial community analyses
supports the shift of the microbial community from one
lacking efficient ARB in the marine hydrothermal vent com-
munity to a community consisting of 80% Geobacter in the
anode biofilm. This demonstrates successful growth and
adaptation of a novel microbial culture for efficient bio-
electrochemical oxidation of butyrate, by combining two
mixed biocatalyst cultures containing species for com-
plementing the desired metabolic steps [77].
Moreover, the use of mixed cultures leads to higher sta-
bility and resistance of MECs and MFCs against the vicissi-
tudes of operational conditions such as temperature, organic
loading rate, etc, which might be possible by a change in
metabolic pathways of microorganisms and predominance of
their community [23,78]. According to the mentioned reasons,
the use of mixed cultures has been recommended for
achieving higher efficiency in bioelectrochemical cells
[11,23,68,78,79].
The SEM micrographs of anodic biofilm presented by Wang
et al. [42], showed the existence of rod-shaped bacteria which
were considered as bioelectrochemically active microorgan-
isms. During two periods (initial and terminal periods,
without and with biohydrogen production respectively), SSCP
analysis showed that while approaching the hydrogen pro-
duction step, Pseudomonas and Shewanella sp. were dominant
species. Also, to investigate the limit of tolerance of biofilm
against imposed tensions, the system was kept in starvation
over one month. Besides biofilm detachment, during this
period, no hydrogen generation had occurred. However, after
this period, by injection of fresh feed, the MEC was recovered
gradually and biohydrogen generation was observed [42].
1481
Dominancy of Pseudomonas and Shewanella were also re-
ported in hydrogen production period by Liu et al. [41]. Isolates
of two MECs were investigated by SSCP and GeoChip (II)
techniques. The first isolate was sampled from the MEC which
had been inoculated by sludge while the second one was
sampled from the MEC which inoculated with the formed
biofilm in the first MEC. The biofilm of the second MEC con-
tained complex organic degradation microorganisms
(including Stenotrophomonas and Lactobacillus); soil microor-
ganisms (including Curtobacterium, Agrobacterium, Fla-
vobacterium and Bradyrhizobium); known exoelectrogens
(including Pseudomonas, Desulfovibrio and Shewanella); and
other microorganisms (including Desulfonauticus, Xenohaliotis
and Marinicola). Also, Geobacter, Shewanella, Rhodopseudomonas
and Desulfovibrio sp. increased during the three months pre-
ceding the beginning of biofilm formation and consequently,
coulombic and hydrogen efficiencies were increased from
zero to 38 and 31 percent, respectively [41].
Wang et al., showed that the existence of suspended mi-
croorganisms was effective for hydrogen production in MECs
[42]. The microbial anode potential (MAP) was analyzed as a
representation of operational factors in a MEC system,
including pH, substrate, applied voltage, etc. For analysis of
microbial communities, the samples were taken from biofilms
and suspended solutions of two MECs, for which the first was
at neutral pH and capable of producing biohydrogen while the
second was at acidic condition (pH < 5) and incapable of
producing biohydrogen. The results showed that all isolates
were very similar in terms of dominant species. Pseudomonas
and Shewanella sp. were both in biofilm and suspended solu-
tion. Also, Stenotrophomonas, Flavobacterium and Agrobacterium
were dominant species, but there was no report on the elec-
tron transport capability of Agrobacterium [42]. This symbiosis
of exoelectrogen microorganisms and other ones forms a
suitable microbial community to consume a wide range of
organic materials and produce more efficient and stable bio-
energy [41,42]. In comparison with the neutral MEC, micro-
organisms in acidic condition were much more abundant in
suspended state and the biofilm of neutral MEC was thicker
and more consolidated than that in acidic condition. The
dominant species in the acidic MEC were Parabacteroides, Lac-
tococcus and Trichococcus sp. of which there was no report on
bioelectrochemical activities [42]. Now, the fundamental
question is what operational parameters are effective on
adhesion of microorganisms to electrodes or cohesion among
different species? Analysis of various microorganisms in pure
and mixed cultures along with the application of different
operational conditions is required to answer this kind of
question which is hoped to be taken into account in future
works.
In a work by Wang et al. [32], an iron-reducing suspended
genus from anodic chamber of a MEC was isolated for the first
time. This species was a gram-negative, short rod, polar fla-
gellum and non-spore forming bacterium. The 16S rRNA
analysis suggested that this strain is a typical fermentative
bacterium and is designated to the strain Bacteroides sp. W7.
The presence of these species increases the probability of
symbiosis of the suspended and other species on biofilm to
attain the higher energy efficiency [32].
1482 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
The study to investigate the effects of various primary
microorganisms' abundance in the inoculum on biofilm for-
mation and MEC efficiency has not been found. Nevertheless,
Aboutalebi et al. [80] investigated the effect of microorganisms
concentration in inoculum on COD removal. The results
showed that rate of COD removal (42 ppm/hr) was maximized
when maximum biomass concentration (6130 mgVSS/L) was
used in inoculum [80]. In addition, Kargi et al. showed that
there initial bacteria concentration was optimum in order to
attain higher hydrogen production in the fermentation pro-
cess [81]. Investigation of this parameter in MECs is needed in
future works.
Despite the results mentioned, it is obvious that there were
many unknown about the types of inoculum (pure and mixed)
and its effect on system efficiency. The questions such as
understanding the competition mechanisms between various
microorganisms during microbial enrichment processes and
after that, the mechanisms of effect of external parameters
such as different mixed cultures and complex substrates on
microbial enrichment and other successive critical questions
still remain vague that response to them can initiate novel
researches.
Effective parameters on microbial communities
Studies carried out on anodic compartment of both MFCs and
MECs showed that anodic biofilm forms from various micro-
bial species which are affected by electrode material, its sur-
face properties and different areas (close to and or away from
membrane) [11,68], operational condition such as system
structure, substrate, additives, inoculum, applied voltage, and
temperature can change microbial diversity [29,34,39].
Effect of anode and applied potentials
It has been confirmed that the change in applied and anode
potentials led to diversification of microbial population
[25,30,82,83]. The energy required by microorganisms (ob-
tained by electron transfer from electron donor to receptor) is
affected by thermodynamic energy of electron donor and re-
ceptor and efficiency of electron transport chain. So, the
anode potential has significant effect on growth and distri-
bution of anode respiring bacteria. Table 2 presents a detailed
list of these correlative studies on MEC systems.
Torres et al. [25] established inappropriate conditions for
growing nonexoelectrogen species to achieve successful
enrichment of anode respiring bacteria. Despite the inoculum
which was taken from wastewater, the anodic biofilm formed
more than 90% of ARBs (that was 97% similar to G. sulfurre-
ducens). They reported higher biofilm growth rate in the lower
Table 2 e The detailed list of related studies on effect of anode/applied potentials in the MECs.
Anode and applied potential range Substrate
Anode potential (0.15  (0.37) vs. SHE) Acetate
Anode potential (0.25  (0.42) vs. Ag/AgCl) Acetate
Applied potential (0.1e1 V) Acetate
Applied potential (0.1e1 V) Synthetic wastewater
Anode potential (0.3  (0.2) vs. Ag/AgCl) Acetate
anode potential (0.15 V vs SHE). In this state, the current
density was 10.3 A/m2 while the current density in the highest
anode potential (i.e. 0.37 V vs SHE) was 0.6 A/m2. Higher
anode potentials led to the formation a thinner and darker
biofilm which was more diverse in microbial species. The SEM
micrographs showed stronger extracellular polymeric sub-
stances (EPS) and nanowires matrix on anodes in lower po-
tentials. In addition, the cyclic voltammetry analysis
confirmed the extreme electron transport by EPS and nano-
wires matrix. In contrast, Karimi Alavijeh et al. [84,85] in
theoretical investigations and Zhang et al. [86] in experi-
mental work have stated that more EPS content makes more
void spaces in the biofilm and as a consequence, this leads to
improper contact between electrogen microorganisms and
the anode surface to transfer electrons. This leads to more
acclimation time for providing the balance between microbial
population rate and electron demand [87,88]. Therefore, with
high EPS content the current density is expected to decrease.
For more clarification, related future researches have critical
role in this part.
In addition, Torres et al. [25] stated that the 16S rDNA
analysis showed the highest microbial diversity on anodic
biofilm of the highest potential (0.37 V vs SHE). About 31% of
the clone sequences were Proteobacteria. The investigation of
microbial communities of the anodes set at potentials of 0.02
and 0.09 V showed a strong selection for Geobacter sp. Ean1
which were 90 and 92% of the sequences, respectively. By
decreasing the set potential to 0.15 V, the community anal-
ysis showed almost a pure culture (about 99%) of the Geobacter
sp. Ean1 [25].
Based on previous work, Commault et al., used low anode
potential and expected a uniform biofilm with dominancy of
Geobacter [34]. However, microbial and electrochemical anal-
ysis revealed high diversity in biofilm in which Geobacter
species were not dominant. The absence of bicarbonate in the
medium, discharge of produced bicarbonate by other micro-
organisms and high ionic strength of the medium was noted
as the reason for this discrepancy. Thus, for perm selection of
Geobacter strains, both mentioned points and anode potential
must be in a suitable situation. They also showed that elec-
trode set at higher voltage led to the onset of energy produc-
tion in a shorter time which is in contrast to Torres et al. [25]
and in agreement to Wang and Wei works [82,89]. In another
part of this study, perm selection of microorganisms at
different anode potential were investigated and resulted in a
decrease in species diversity and it was observed that special
species (which were very similar to Geobacter psychrophilus)
were dominant. Another significant result of this study was
the use of ARISA analysis for the first time to demonstrate that
closely related strains of Geobacter showed very different
competition capabilities at different anode potentials. For
Studied features References
Perm-selection of ARBs [25]
Different competitive advantages of microbial species [34]
Diversity and viability of biofilm [33]
Enzymes activity and microbial metabolism [90]
Biocatalytic activity of biofilm in different MEC designs [30]
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
example, G. psychrophilus was dominant in biofilm formed at
0.25 V vs. Ag/AgCl while at 0.36 and 0.42 V vs. Ag/AgCl, G.
sulfurreducens were dominant. Moreover, power density in this
state (250e270 mW/m2) was 30 times higher compared with
the state in which Geobacter species were not dominant [34].
Investigation of applied potential (in range of 0.1e1 V) on
microorganism populations in biofilm showed that variation
in this parameter can cause major changes in microbial spe-
cies and their viability. Chae et al. [33] investigated the mi-
crobial community of MFC (MEC without applied voltage) with
16S rRNA and then by exerting applied voltage and converting
the MFC to MEC, the microbial community was analyzed
again. The results showed that MFC microbial community was
very diverse and Proteobacteria, especially b-Proteobacteria
(51.2%) was dominant. Increasing the applied potential grad-
ually reduced microbial diversity and d-Proteobacteria (72%)
was eventually dominant. In addition, increasing the applied
potential reduced cell density but increased viability (i.e. the
number of active microorganisms to inactive ones) [33].
A change in applied potential can also influence enzymes'
activity and as a result, change microbial metabolism. Mohan
et al. [90] showed that change in applied potential can affect
the activity of dehydrogenase enzyme and consequently
affect substrate degradation rate (SDR), volatile fatty acid
(VFA) generation and biohydrogen production. The activity of
dehydrogenase enzyme and biohydrogen production were
maximum at applied potentials of 0.6 and 1 V, respectively
[90].
Kumar et al. [30] investigated anode potential influence on
biocatalytic activity of biofilm in different MECs designs
(including single chamber and two chamber MECs). The
maximum current density in single chamber MEC was ob-
tained at the highest anode potential (i.e. 0.2 V vs. Ag/AgCl)
[30]. This observation has also been revealed in other work on
a single chamber MEC [91]. SEM micrographs of the biofilm
formed at 0.2 V vs. Ag/AgCl showed that it was thicker and
more densely packed than the ones at the more negative
applied potentials (for instance at 0.3 and 0.2 V vs. Ag/
AgCl). On the other hand, when the same experiments were
conducted in membrane separated electrochemical (dual
chamber) cell configuration, the more negative applied po-
tentials produced higher current densities and thicker and
more densely packed biofilms, compared to anodes polarized
at the most positive applied potentials. So, influence of anode
potential on biofilm growth and its biocatalytic activity
strongly changes considering the system configuration. It
should be noted that in any configuration, DGGE analysis
showed that more positive anode potential led to more di-
versity in microbial species. Besides, the composition of mi-
crobial species was different in different configurations [30].
As Table 2 shows, the effect of anode and applied poten-
tials on microbial communities were investigated on biodeg-
radation of acetate in MECs except Mohan et al. work [90].
However, the question remains that what are the major
changes in microbial community of MEC for biodegradation of
complex substrate in different anode and applied potentials
and consequently, does the applied potential change the rate
of sequence reactions of complex wastewater biodegradation?
The responses to these questions are needed in future studies.
1483
Substrate
It is obvious that the kind of substrate used has inevitable
effects on microbial community. In addition, the used sub-
strate can during the transformation of a MFC to a MEC, in-
fluence the diversity of microbial species. For example, using
acetate as substrate in a MEC led to decrease in microbial di-
versity and increase in the number of G. sulfurreducens in
comparison to MFC [35]. Characterization of the biofilm of the
acetate-fed MEC showed that Geobacteraceae. Pelobacter pro-
pionicus clones which was made up 54% of the community,
and G. sulfurreducens which accounted for 38% of the popula-
tion, were dominant species. Using potato wastewater as
substrate, there was no change in Geobacteraceae population
when the MFC were converted into the MEC, but the number
of G. sulfurreducens clones decreased to about 62% and the
population of G. metallireducens clones increased (from about
zero to 14%). Kiely et al. also reported that dairy manure-fed
MEC had relatively few Geobacteraceae (<12%) and did not
observe any hydrogen gas production. They noted that the
presence or absence of Geobacter does not explain the failure
of the biofilm to produce current in the MEC, when power was
successfully generated by the same biofilm in the MFC. They
hypothesized that oxygen utilization may have been critical to
the functioning of the microbial community with the dairy
manure [35].
Cusick et al. [36] investigated the effect of using winery and
domestic wastewater as substrate on MEC microbial com-
munities. The microbial community in the domestic
wastewater-fed MEC was dominated by four groups of Geo-
bacter strains (52.6% of the community), including G. metal-
lireducens, G. sulfurreducens, Geobacter lovleyi, and Geobacter
uraniireducens. The MECs which fed with winery wastewater
was dominated by G. sulfurreducens and Roseivivax, made up 44
and 14% community, respectively. The presence of Pelobacter
propionicus, as a fermentative member of the Geobacter species
family, has been confirmed in all MECs by 16S rRNA analysis
which is an emphasis on symbiosis and contribution of elec-
trogenic and fermentative species in organic material of
wastewater removal and bioenergy production [36].
Thygesen et al. [38] also investigated the effect of using
hydrolysate as a complex mixture of polysaccharides on MEC
microbial community and biohydrogen production. The mi-
crobial community analysis was conducted by DGGE and
compared with microbial community of acetate-fed MEC.
Biodegradation of 96% of extracted hemicellulose in the hy-
drolysate fed-MEC showed that hydrolysate is an appropriate
substrate for bacterial growth. For acetate fed-MEC, the
number of bacterial species in anodic biofilm and anolyte
were 14 and 18 respectively; while for hydrolysate-fed MEC,
they were 17 and 22, respectively.
Moreover, acetate-fed MEC had biofilm microbial commu-
nity with microbial richness of 9 and 26 in the biofilm and
anolyte solution. These values when the MEC was fed with the
hydrolysate increased to 40 in the biofilm and 56 in the anolyte
solution. It should be noted that when acetate was used as a
substrate, 69% of microbial species in the biofilm and the
anolyte solution was similar while for hydrolysate fed MEC
this value reduced to 56%. Complexity of hydrolysate in
1484 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
comparison to acetate led to decrease of the Co value (more
even microbial community) both in the anolyte solution and
the biofilm. G. sulfurreducens was the dominant species for
both substrates. Microorganisms phylogenetically correlated
to Ruminobacillus xylanolyticum, Bacteroides graminisolvens, Pal-
udibacter, Cloacibacillus evryensis, and Clostridium coccoides were
identified only when hydrolysate was used as a substrate in
the MEC [38].
Ruiz et al. [46] investigated the effect of different initial
concentrations of a mixture of acetic and propionic acids as
common products of dark fermentation on MECs microbial
ecology. The quantitative-PCR analysis showed that Proteo-
bacteria, Firmicutes and Bacteroidetes and Arcobacter were the
dominant genera. In addition, they found that some other
bacteria like Arcobacter, Desulfomicrobium, Desulfobulbus, Pseu-
domonas, Macromonas and Azovibrio were the most represen-
tatives and could be associated with hydrogen production in
MECs. The results showed that although defined acetic and
propionic acid concentrations fed affected the structure of the
microbial consortia that developed at the anode, the initial
inoculum played a major role in the development of MEC
microbial consortia [46].
It is obvious that there are many unknown about the ef-
fects of various substrates on microbial communities in MECs
and there are a few studies in this field [35,36,38,46]. Moreover,
except for Ruiz et al. [46] study, there are no investigation on
the effect of substrate concentration, its ingredient and also
the use of more complex substrates on microbial population
and species competitiveness.
Additives
Low rate of electrons transfer from microorganisms to anode
and from cathode to the final electron receptors have always
been one of the most important issues regarding efficiency
improvement in bioelectrochemical systems. In this regard,
effect of additives in anodic and even cathodic chambers such
as Tween 80 [27,92], (conductive, non-conductive or semi-
conductive) iron-oxide nanoparticles [27,29,93], and eDNA
[27] have been investigated. For example, addition of Tween 80
to MFC led to an increase of about nine times the power
density [92]. Iron-oxide nanoparticles additive resulted in 30
fold enhancement in the produced current [93]. Addition of
these materials, on the other hand, can change growth rate,
stability, morphology and structure of biofilm [27] and also
COD removal rate and system stability at different acidity [29].
Ren et al. [27] examined whether several chemicals (eDNA,
Tween 80 and Fe(OH)3) would affect the formed biofilm in a
single chamber MEC. The SEM micrographs showed that
addition of Fe(OH)3, despite no noticeable effect on anodic
biofilm, led to formation of a highly porous structure on
cathodic biofilm and consequently caused considerable in-
crease in current density (from 6.1 to 8.8 A/m2) [27].
Effect of Fe(OH)3 addition on biofilm morphology was
investigated by Zhang et al. [29]. TEM micrographs from
sludge samples showed that a great amount of flocculus EPS
(which is principally composed of exopolysaccharides with
microchannel for fluid movement [58]) adhered to the surface
of species. EPS preferred to bind with divalent metals such as
Fe2 to form a stable structure, thereby assisting the
enrichment of effective microbes. Pyrosequencing and DGGE
analysis revealed that this additive also increase the richness
of dominant bacteria and archaea communities and species
diversity in the biofilm [29].
Physical and chemical characteristics of electrodes
Since electrodes are platforms for biofilm growth and accli-
mation, their physical and chemical characteristics can in-
fluence microbial activity, and by extension system efficiency.
For instance, Xu et al. [94] used an iron nanoparticle-decorated
anode to investigate electrode modification on biocatalysis
activity of Shewanella oneidensis MR-1 in a MEC. The results
obtained from MEC with a modified electrode compared with
the same MEC with plain graphite anode, showed that the
modification led to an increase in expression of genes related
to nanowires, flavins, and c-type cytochromes indicating an
enhancement of electron transfer to the anode. This has
contributed to a significant increase in current density which
was more than 5.9 fold of current density in the MEC with
plain graphite [94].
Comparison of microbial communities formed in
different electrodes is another subject that revealed the sig-
nificance of electrode characteristics in the formation of
microorganism population. The study of Selembo et al. is the
only work in this subject. Selembo et al. [95] showed that the
use of nickel powder constructed cathode as a cost effective
alternative to platinum did not have an influence on anodic
biofilm. The closest matches to sequences obtained from the
anode biofilm from the MEC with a nickel cathode were
Pelobacter propionicus (54.3%) and G. sulfurreducens (21.7%)
which was similar to what was obtained with platinum. The
initial contemplation of Selembo et al. study showed that
biocatalyst communities were the same in the two different
electrodes. However, it seems that comparison of carbon
based and metal based electrodes in respect of their micro-
bial community in MECs should be considered in future
researches.
Temperature
The study of Lu at al [43] was the only work in which effect of
temperature on MEC microbial community had been inves-
tigated. They investigated microbial community formed for
biodegradation of glucose in a MEC at two temperatures of 4
and 25  C. The high similarity of CV analysis results to that of
pure Geobacter species showed that (i) the dominant species
were the microorganisms belonging to the genus Geobacter,
or that they were strongly related to it (ii) the outer mem-
brane redox proteins (cytochromes) and the electron transfer
mechanisms were identical or at least very similar to those of
the exoelectrogens. Analysis of GS-FLX pyrosequencing of
the 16S rRNA gene 454 on MECs isolates in different tem-
peratures showed a decrease in microbial diversity when a
temperature of 4  C is used, this could be attributed to
reduction in mesophilic and/or thermophilic species rich-
ness compared to the original inoculum. Six and nine bac-
terial phyla were identified in communities of MEC at 4 and
25  C respectively. The similarities of phyla, however, were
very high and the total observed phyla in both MECs was ten.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
At the phylum level, Bacteroidetes, Proteobacteria and Firmi-
cutes phyla were dominant in both temperatures. In respect
of the dominant class levels, two communities still the
relatively similar structure, and the majority of sequences
belonged to classes of g-proteobacteria, d-proteobacteria, Bac-
teroidia and Clostridia. Despite the fact that 20 classes were
observed at 4  C, 88.4 and at 25  C, 83.5 percent of species
were related to 4 stated classes (including g-proteobacteria, d-
proteobacteria, Bacteroidia and Clostridia) and 3 mentioned
phyla (including Bacteroidetes, Proteobacteria and Firmicutes
phyla). Contrary to these similarities, analysis at genus level
showed remarkable differences between two communities.
At 4  C, Dysgonomonas (which is composed 36.6% of the
community) while at 25  C, Bacteroides (which is composed
21.5% of the community) were the most dominant genera in
the MECs isolates. As CV analysis predicted, Geobacter sp. was
the most predominant known exoelectrogen in both bacte-
rial communities [43].
Methanogens and their controlling methods
One of the most important matters in symbiosis of different
microbial groups that must be noticed is that the existence of
other microorganisms among exoelectrogen ones may have
an effect on electricity and hydrogen production negatively
[39]. As previously mentioned, the basic difference between
MFCs and MECs is completely anaerobic in condition (whether
in anodic or cathodic chamber) in MECs. This anaerobic con-
dition can cause an undesirable increase in species growth
specially methanogen microorganisms such as Methano-
brevibacter ruminantium, Methanosarcina mazei and Meth-
aomicrobium mobile [59].
In MECs, methane is typically produced in two reaction
pathways [61,96]. In each pathway, the special groups of
methanogens play critical roles as biocatalysts. Acetoclastic
methanogens such as Methanosaeta and Methanosarcina [96] in
the presence of acetate convert it to methane as seen in re-
action (1). In addition, production of methane from hydrogen
and carbon dioxide can be achieved by hydrogenotrophic
methanogens such as Methanobacteriales [26,96,97] as shown
by reaction (2):
Acetoclastic Methanogens
C2 H4 O2 !CH4 CO2
Hydrogenotrophic Methanogens
4H2 CO2 CH4 2H2 O
! (2)
These lateral reactions resulted in a decrease in coulombic
and hydrogen efficiency. For example, in a MEC, an increase
in methane production during 86 days from 9 to 116 mmol led
to an increase in electron loss and a decrease of hydrogen
recovery from 2.2 to 29% and from 77 to 50%, respectively
[96].
Procedure for these undesirable reactions can be changed
with respect to operational conditions. For instance, in a
digester, 70% and 30% of methane were produced through the
first and second reaction pathways respectively [98]. Wang
et al. [61] showed that when acetate concentration was high,
little methane gas was generated during the initial reaction
time (<12 h) in a fed batch MEC while most methane was
1485
produced at the end of a batch cycle when hydrogen and
carbon dioxide gases were at the highest concentrations.
Therefore, hydrogenotrophic methanogens were noted as
mainly responsible for methane production. In addition,
increasing the batch time from 24 to 72 h resulted in complete
conversion of hydrogen gas to methane [61]. In contrast, Chae
et al. [96] reported that methane production is in control of the
acetoclastic methanogens activity. However, there is no study
on competition of methanogens groups and its effective pa-
rameters which are hoped to taken into account in future
works.
The ways to reduce methanogens activity
In advent of an issue, the first step is to identify the problem
and its governing peripheral characteristics. Since the activity
of methanogens is one of the most important problems in low
efficiency of bioelectrochemical systems, specially MECs [39],
the basic approach of most works is to study methanogen
species and their characteristics comprehensively.
Until now, various methods in controlling methanogens
have been presented. Already used methods in elimination or
inactivation of methanogen species are listed in Table 3.
However, the coupled systems (including both MEC and
anaerobic digesters) were used in some studies for cogenera-
tion of hydrogen and methane [59,61,62,96].
In MFC-MEC coupled systems, exchanging the electricity-
assisting and hydrogen producing function between MEC
and MFC, as confirmed by FISH analysis, reduced the activity
of methanogens effectively. The use of this method led to an
increase by 29% in the volume of hydrogen produced [101].
Moreover, moving from batch to continuous mode decreased
methane production considerably [91].
Table 3 e The used methods in elimination or
inactivation of methanogen species.
Used method References
Using pure culture instead of mixed [31,99]
culture
Using alternative buffers instead of [1]
bicarbonate
Exposure of electrodes to oxygen in a [46,100]
(1) buffer solution before each batch cycle
Exchanging the electricity-assisting and [101]
hydrogen-producing function between
MEC and MFC
pH reduction [38,91,96,99]
Operating at psychrophilic condition and [43,62,96]
temperature shock
Reduction of MEC run time [26,59,102e104]
Operating in more positive anode and [61,91,102,103,105e107]
applied potential
Using perm-selective membrane in double [108]
chamber MEC
Addition of chemicals such as BES and [40,44,96,109]
lumazine
Pretreatment of electrodes before MEC run [99]
Replacing the anode into a new MEC [91]
Pretreatment of the MEC influent to [110,111]
eliminate undesirable species
Modification of MEC design [26,106,112]
1486 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
Lu et al. [62] investigated the effect of temperature on
methanogens population. Methanogens were inhibited by
operating the acetate-fed single chamber MEC at 4 and 9  C. At
these temperatures, dominant species shifted from Geobacter
chapelleii to G. psychrophilus and the growth of hydro-
genotrophic methanogens such as Methanobrevibacter arbor-
iphilus which are recognized as the most important
microorganisms for methane production in MECs [113], was
effectively inhibited under psychrophilic conditions [62]. The
abrupt decrease in temperature (from 32 to 20  C), however,
had no effect on methanogenesis activity and MEC coulombic
efficiency [96].
In addition to temperature shock, effect of acidity shock,
exposure to oxygen and effect of addition of specific chemical
on methanogens activity were investigated. The results
showed that the pH shock from 7 to 4.9 had no effect on
methane production. However, Nam et al. [91], showed a
decrease in pH reduced both methanogens and exoelec-
trogens activity [91]. Similarly, oxygen exposure reduced the
activity of methanogens (which are obligate anaerobes) and
other beneficial obligate anaerobes in electrons production
such as Geobacter sp. which consequently had no effect on
coulombic efficiency [96]. Moreover, Folusho et al. [100] re-
ported that electrode exposure to air (for 24 h) and dissolved
oxygen in the buffer solution (for 120 h) did not have any effect
on methanogenesis activity. The main reasons for metha-
nogens resistance to oxygen exposure were explained as fol-
lows [96]:
 protection of methanogens from oxygen by biofilm matrix
 the presence of facultative microorganisms and estab-
lishment of the anaerobic microenvironments
 the presence of oxygen tolerant species of methanogens.
Addition of chemicals such as BES and lumazine had an
interesting effect on methanogens inactivity. The use of BES
led to not only a considerable reduction or even cessation of
methane production, but also resulted in an increase in
overall hydrogen efficiency from 56 to 80% [96]. The hydrogen
volume, in the presence of BES, was 1.4 times more than the
control mode. It must be said that the inhibitory influence of
BES was retained even after 10 batch cycles with no further
BES addition. In contrast to BES, lumazine did not have any
effect on methane production [96]. Some works showed that
lumazine had influence only on hydrogenotrophic metha-
nogens [114,115] which convert the back diffused hydrogen
from cathodic chamber to methane. The weakness of luma-
zine on methanogens activity in previous studies contributed
to low rate of the hydrogen back diffusion to anolyte, which
led to hydrogenotrophic methanogens which were not
dominant to prevent their activity by lumazine in the MEC
[96].
Besides BES and lumazine, special antibiotics including
neomycine sulfate, 2-chloroethane sulfonate, 8-aza-hypo-
xanthine were examined and showed that hydrogen effi-
ciency of MECs with these additives could be improved
through inhibition of methanogens' activities [109].
Methane production in cathodic chamber is another
problem of interest which was investigated by Chae et al. [96].
They reported that methane production in cathode
compartment was very low (<3.5% of the produced hydrogen)
but its origin remains unclear. For better understanding of the
origin of methane in cathodic chamber, BES was used in
anodic chamber to reduce anodic methane production and
eliminate concentration gradient and to demonstrate that the
diffusion of methane from anodic chamber could not be the
reason of cathodic methane presence. PCR analysis confirmed
the presence of acetoclastic and hydrogenotrophic metha-
nogens in cathodic chamber, but as long as there was no ev-
idence of acetate in it, the use of BES and even autoclaving of
cathodic chamber did not terminate methane production.
Thus, other ways including direct conversion of an electrical
current to methane (reaction 3) and catalytic conversion of
hydrogen to methane (reaction 4) was responsible for
methane production in cathodic chamber [96]. However, more
studies are required for better understanding.
CO2 8H 8e ! CH4 2H2 O (3)
4H2 CO2 ! CH4 2H2 O (4)
Some studies reported that methanogens' activity
decreased in the more positive anode and applied potentials
[61,91,107,116]. So, one can say that more input energy led to
shorter cycles and therefore less methane production [61,91].
For example, a change in anode potential from 0.4 to 0.2 V
led to an increase in input energy and decrease in MEC batch
time from 40 to 8 h and consequently methane production
reduced by about 90% [91].
Pretreatment of electrodes is another way to reduce
methanogens activity. Hrapovic et al. have developed gas
diffusion cathodes with electrodeposited nickel (Ni) particles.
The modified cathodes facilitate hydrogen transport through
the gas diffusion layer, so the loss of hydrogen production to
methane was at minimal level (less than 5% of the off gas)
[112].
Modification of MEC design contributed to the prevention
of methane production. For instance, the MEC constructed by
Guo et al. [106] resulted in reduction of methane production
and improvement of hydrogen efficiency. Placement of cath-
ode on top of anodic chamber prevented hydrogen entrance to
anodic chamber and therefore resulted in a decrease in
methane production [106]. Similarly, Lee et al. [26] fabricated
an up-flow single chamber MEC in which the cathode was
placed on the top of the MEC. Quantitative real-time PCR
analysis of the MEC isolates showed that Geobacteraceae was
composed of 41% of the total bacteria in the anode's biofilm,
whereas they were only 7% in the cathode's biofilm. The
acetoclastic methanogens such as Methanosaetaceae and
Methanosarcinaceae were not observed remarkably in both
anodic and cathodic isolates due to the suitable MEC design,
resulting in shorter cycle time (below 34 h). However, hydro-
genotrophic Methanobacteriales were present in both anodic
and cathodic biofilms. By increasing acetate concentration
which consequently increased MEC run time, significant
methane was produced. However, the reduction of coulombic
efficiency due to enhancement of acetate concentration was
negligible; so decrease in hydrogen yield was as result of lower
cathodic conversion efficiency. It can be implied that hydro-
genotrophic methanogenesis was mainly responsible for
methane production. Besides, the acetate oxidizing ARB out-
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
competed acetoclastic methanogens for acetate consumption
[26].
Most of the methods used to reduce or eliminate biological
methane production in the aforementioned studies were
evaluated in a single chamber cubic MEC by Rader et al. [117].
It was reported that the application of high voltage (0.9 V) and
exposure to oxygen (for 24 h) did not have any effect on
methane production. There was prolonged addition of BES to
inoculation medium until its presence in the medium reduced
the methanogens' activity. After five fed-batch cycles, by
dropping BES concentration, the biological production of
methane was intensified. In addition, temperature reduction
from 30 to 20  C led to a 40% decrease in methane production
and also 15% reduction in hydrogen production [117].
Besides methanogens, there are other hydrogen scavengers
such as homoacetogens [39], denitrifiers (nitrate reducing
bacteria) [118,119] and sulphate reducing bacteria (SRB)
[58,120]. The activity of the two latter groups can be inhibited
by exclusion of related electron acceptors (including nitrate
and sulphate) [39]. Homoacetogens produce acetate from
hydrogen and carbon dioxide [121]. But when hydrogen was
used as substrate and other hydrogen dissipaters were
inhibited, symbiosis of homoacetogens and exoelectrogens led
to efficient consumption of hydrogen and current densities
similar to acetate-fed biofilm anodes (10 A/m2). It should be
noted that this was endorsed in the double and single chamber
MECs. The presence of dissipater species is thus an effective
factor for hydrogen loss. The 16S rRNA and FTHFS analysis
showed that in a double chamber MEC where hydrogen was
used as substrate, concentration of suspended homoacetogens
in anolyte were remarkably higher (1000 times) than in the
attached form in the biofilm isolates. In the anolyte, 57% of the
clones were from the genus Desulfovibrio as dominant species
and there was no trace of Geobacter, Actinobacteria, or Bacter-
oidetes species. It also showed that the ratio of Geobacteraceae
to other microorganisms in the biofilm was more than 100 fold
of this ratio in the anolyte. The low ratio of Geobacteraceae to
other species in the anolyte conforms to the fact that ARB must
be associated with the anode biofilm. Moreover, the use of
hydrogen as substrate led to a drop in predominance of G.
sulfurreducens from 95% in acetate-fed biofilms to 71e77% for
the hydrogen-fed biofilms [25,39]. It also resulted in an increase
in Actinobacteria, Dysgonomonas, homoacetogens and other
acetogenic bacteria (including Acetobacterium, Eubacterium, and
Spirochaeta) in the biofilm [39]. It should be noted that homo-
acetogens holds promise for high electron recoveries in a
separate anode chamber and they could potentially account
for hydrogen losses in a single-chamber MEC.
Gao et al. showed syntrophic interactions among fermen-
ters, homoacetogens and ARBs would allow MECs to maintain
high current density and coulombic efficiency [122]. The
establishment of syntrophic interactions of fermenters and
ARBs in the MEC was speculated by a large reduction of par-
ticulate matters, accumulation of propionate, and trivial
concentration of acetate in the effluent. Besides, the detection
of Thermanaerovibrio spp., which are fermenters and capable of
using simple acids and hydrogen gas [123], implied that these
species play critical role as syntrophic partners with ARBs like
Pelobacter, homoacetogens, or H2-utilizing ARBs to facilitate
volatile fatty acids (e.g., propionate) fermentation in the MEC.
1487
The use of fermentable substrates led to a fairly large
reduction in coulombic efficiency of microbial electrochemical
cells [124,125]. This contributed to more hydrogen production
by using fermentable substrates (such as butyrate, propionate,
and lactate) and the more competencies of methanogens in
consumption of this hydrogen in comparison to exoelec-
trogens. Generally, the most important substances which, in
absence of the exterior electron receptor (such as oxygen, ni-
trate and sulphate) inhibit current generation from electrons
are methane, biomass and soluble substances produced by
microorganisms. Parameswaran et al. [44] investigated ethanol
consumption (as fermentable substrate) in a MEC to explore the
relationship between ARBs and other microorganisms. The
results revealed that coulombic efficiency was a function of the
interactions and competition among ARBs, methanogens and
fermentative species. The coulombic efficiency, in the presence
of methanogens, was about 60% and 26% of the generated
electrons converted to methane by methanogens. QPCR anal-
ysis showed that the only methanogen genus was Meth-
anobacteriales which belongs to hydrogenotrophic group and
there was no trace of acetoclastic species. Thus, ARBs are more
competent in the consumption of acetate in comparison to
acetoclastic methanogens. It is also confirmed by the acetate
concentration needed to reach to half of the maximum rate of
ARBs and acetoclastic methanogens. They also showed that
Geobacteraceae were the most prominent family in anodic bio-
film. The addition of BES to MEC in addition to reduction of
suspended archea in both anolyte and biofilm led to cessation
of methane production and increase in coulombic efficiency to
84%. Thus, Parameswaran et al. showed that the inhibition of
methanogens activity and growth, in MECs with fermentable
substrates led to interaction and competition among different
species and resulted in more current production. Moreover,
since the ARBs do not have the ability to consume ethanol, the
current increased after conversion of ethanol to acetate and
acetate consumption by ARBs [44].
Parameswaran et al. [40] following their previous study,
investigated the anolyte and biofilm microbial community in
methanogen inhibition and control conditions with more de-
tails. They showed that in the control condition (no BES added),
the produced hydrogen was lost by hydrogenotrophic metha-
nogens and on the other hand by addition of BES to inactivate
hydrogenotrophic methanogens, the produced hydrogen was
converted to acetate by homoacetogens which then led to
electricity generation. Microbial analysis on anolyte and anodic
biofilm revealed that the suspended archaea population, in the
case of control condition, was more prominent in comparison
to its population in anodic biofilm. In this condition, about 92%
of the total bacterial 16S rRNA genes in the anolyte belonged to
the phylum Proteobacteria, of which 86% of this population was
Deltaproteobacteria. The predominant genus among Deltaproteo-
bacteria was Pelobacter (ethanol-fermenting bacteria). Epsilon-
proteobacteria formed the remaining 6% of the Proteobacteria
phylum. Similarly, Proteobacteria were prominent but more
diverse in the anodic biofilm than in the anolyte being distrib-
uted among Deltaproteobacteria (38% of Proteobacteria), Epsilon-
proteobacteria (31% of Proteobacteria), and Betaproteobacteria (7%
of Proteobacteria). By addition of BES to inactivate hydro-
genotrophic methanogens, the ratio of archaea to total bacteria
in both anolyte and anodic biofilm reached to below 0.3%. In
1488 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
this condition, Proteobacteria and Pelobacter was the prominent
phylum and genus respectively in both anolyte and biofilm
isolates. The FTHFS showed the presence of homoacetogens in
only methanogen inhibition condition [40].
Lu et al. [43] investigated the activity of methanogens at 4
and 25  C. There was no methane production at 4  C. So, the
activity of methanogens can be decreased at lower tempera-
tures. However, cathodic hydrogen recovery was low (61e68%)
which was attributed to longer batch cycle time, reduction of
current and hydrogen evolution reaction rate by decreasing
temperature. Meanwhile, the low cathodic hydrogen recovery
at 25  C was because of hydrogen conversion to methane.
There was no presence of homoacetogens at 4  C and most
contribution was between fermentative and exoelectrogen
microorganisms. In this condition, glucose was fermented by
fermentative species and then, due to inactivity of homoace-
togens and methanogens, exoelectrogens consumed the pro-
duced acetate without any competitor to generate bioenergy
[43].
Dhar et al. [110] used AnMBR (Anaerobic Membrane Bio-
Reactor) pretreatment on substrate, which led to entry pre-
vention of fermentative and methanogen microorganisms to
terminate methane production. Comparison of COD removal
with the raw and pretreated substrate in MECs revealed that
pretreatment led to elimination of some beneficial microor-
ganisms like propionate fermentative and therefore rate of
COD removal by ARBs decreased and propionate concentra-
tion at outlet increased. Another significant result in this
study is rise of the coulombic efficiency to above 100% which
can be attributed to the hydrogen back diffusion from
cathodic to anodic compartment and reconversion of it to
electrons (by ARBs) or acetate (by homoacetogens species),
and/or the consumption of soluble substances which are
produced by other microorganisms by ARBs. In addition, the
consumption of intracellular biopolymers such as PHB may
have contributed to this result. However, the consumption of
intracellular biopolymers was reported as the main reason for
the observed coulombic efficiency. Moreover, biofilm analysis
by fluorescent microscopy confirmed the presence of
biopolymers-accumulating microorganisms [110].
Hu et al. [99], for the first time, investigated hydrogen
production in membrane less MECs using Shewanella oneidensis
MR-1 and mixed culture. By using mixed culture, higher cur-
rent density and hydrogen production rate were acquired.
They used three methods for methane production inhibition.
The first was pH reduction from 7 to 5.8 which had no inhib-
itory effect. Cathode exposure to air, during medium
replacement, for 15 min led to reduction in the produced
methane below 1%. The third method was placement of the
anode prior to transfer from the MFC to the MEC in 100  C
water for 15 min. The high temperature led to extreme dam-
ages to most microorganisms specially exoelectrogens, so
hydrogen production decreased significantly and metha-
nogens elimination was not complete [99].
The alkaline pretreatment of raw wastewater showed sig-
nificant impact on enhancement of methanogens activities.
Sun et al. [111] pretreated raw wastewater by specific alkaline
dosage of 1.16 0.02 g NaOH per gram of volatile suspended
solids (VSS) to adjust the pH to 9.68. The methane production
was increased by 1.5 fold compared with MECs fed with raw
waste activated sludge as substrate. The community analysis
showed that the dominance of Geobacter in MECs, which is a
well-known exoelectrogens contributed to the high current
generation. The analysis of archeal genera showed the domi-
nancy of Methanocorpusculum for MECs with more composition
for pretreated-fed MEC. The dominance of methane producing
archeal Methanocorpusculum showed that methane production
was achieved via hydrogenotrophic methanogens [113].
In spite of several research works carried out in this area,
the complementary study of methanogens behavior with
complex substrates under different applied potentials and
also hydrodynamic effects on methane production in the
continuous MEC or even investigation of another additives for
the elimination of methanogens are subject which are hoped
to be taken into account in future works.
Biocathodes in MECs
In order to reduce fabrication costs of microbial electro-
chemical cells which is a major challenge in commercializa-
tion of these systems, biocathodes are common alternative to
expensive catalytic cathodes like platinum [1,56,64,66]. These
catalysts, besides being expensive, have another problems
such as environmental concerns during mining and extrac-
tion processes and its high probability to be poisoned by
hazardous chemicals such as sulfide [59].
In biocathodes, microorganisms catalyze cathodic half re-
actions (i.e. hydrogen evolution reaction) and due to significant
reduction in MECs fabrication cost in comparison with com-
mon catalysts, and besides self-renewability and sustainabil-
ity attract particular interest in bioelectrochemistry studies
[1,64]. On the other hand, produced current densities in MFCs
and MECs are 3 orders of magnitude lower than those of con-
ventional fuel cells. Thus, the use of such an expensive mate-
rial as cathode catalyst is not justified in these systems [1].
Rozendal et al. for the first time investigated the use of
biocathodes in MECs [1]. The process of biocathode formation
was carried out in three steps: (i) formation of an acetate and
hydrogen oxidizing bioanode after inoculation with a mixed
culture of electrochemically active microorganisms (ii) adap-
tation to hydrogen selective oxidation species, and (iii) polar-
ity reversal to hydrogen producing biocathode and adaptation.
The results showed that the use of biocathode resulted in 8
fold enhancement in hydrogen production rate (about
0.63 m3/m3d) in comparison to control electrode [1].
There are only a few studies on the investigation of micro-
bial community of biocathodes. Croese et al. [63] carried out for
the first time a study on biocathodic microbial community. 16S
rRNA and DGGE analysis showed that Desulfovibrio, Firmicutes
and Bacteroidetes were dominant in biocathode [63].
Fu et al. [64] examined the potential of thermophilic mi-
croorganisms as biocatalysts in the cathode of MEC. The used
biocathode had been transferred from a single chamber MEC
to a double chamber one. The use of biocathode led to a
considerable increase in current density and hydrogen pro-
duction rate. The LSV analysis indicated that although
hydrogen production with thermophilic biocathode is per-
formed at lower potential (0.62 V vs. SHE) in comparison to
Pt-based cathode (0.4 V vs. SHE), it was higher than
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
mesophilic biocathode and stainless steel mesh cathodes. 16S
rRNA showed that there was six different phyla, in which
Firmicutes (137 clones, 77.4% of community) followed by Cop-
rothermobacter (35 clones, 19.8% of community) was the
dominant phylum. It should be noted that in contrast to re-
ported mesophilic biocathodes [63], there was no trace of
Proteobacteria, in the thermophilic biocthaode [64]. In addition,
their results showed that deactivation of microorganisms in
biocathode in the presence of carbon monoxide, was evidence
of hydrogenase activities (such as Desulfovibrio caledoniensis,
Desulfovibrio paquesii, Desulfovibrio sp. G11, Desulfitobacterium,
G. sulfurreducens) which led to the production of hydrogen in
the control condition.
The presence of Firmicutes as dominant species in small
setup of MECs was reported in the work of Croese et al. [126].
They indicated that the design of MEC had a significant effect
on the composition of the biocathode biofilm. This effect was
more considerable than the effect of differences in carbon
source (autotrophic or heterotrophic). In this way, Proteobacteria
were enriched on the cathodes in the large scale MECs [126].
Villano et al. [56] employed hydrogenotrophic dechlorina-
tion of bacteria including Desulfitobacterium and Dehalococcoides
as biocathode microorganisms for catalyzing the hydrogen
evolution process in the presence and absence of methyl viol-
ogen as a redox mediator. The Desulfitobacterium enriched cul-
ture was capable of catalyzing hydrogen production without
the redox mediator at cathode potentials lower than 700 mV
vs. SHE. In the presence of the mediator, hydrogen production
was observed for both cultures with the cathode potential set at
450 mV vs. SHE. The use of Dehalococcoides culture led to an
energy efficiency that was 1.7 times higher than that of Desul-
fitobacterium culture. Moreover, hydrogen production was slight
in abiotic electrode (without biocathode microorganisms) [56].
Sulphate reducing bacteria (SRB) which often exists in the
anaerobic environments such as soil and marine sediments,
are other species used as biocathode by Martins et al. [55].
These prokaryotic bacteria used sulphate and sulphure as final
electron receptor. Also, some of this group such as Desulfovibrio
desulfuricans ATCC 27774 can reduce nitrate [58]. It should be
noted that hydrogen has a very basic role in metabolic path-
ways of Desulfovibrio. In some cases, hydrogen was consumed
by sulphate respiration simultaneously as well as it can be
produced in absence of sulphate [58,120,127e130]. Lojou et al.
[129] investigated the production and consumption of hydrogen
by Desulfovibrio desulfuricans and showed that the use of methyl
viologen resulted in maximization of current generation [129].
It was reported that the formed biofilm which was inoculated
by Desulfovibrio caledoniensis reduced the overpotential to about
0.2 V. The current generation and hydrogen oxidation by this
biofilm were achieved at applied potential of 0.24 V (vs. SHE).
The results showed that the biocatalysts in biofilm were also
able to obtain electrons from the 0.61 V (vs. SHE) polarized
electrode to form hydrogen [130].
Aulenta et al. [131] showed the capability of Desulfovibrio
paquesii in direct electron conduction and hydrogen con-
sumption or production in which the coulombic efficiency
reached about 100% [131]. Martins et al. [55] used SBR species
identified as Desulfovibrio vulgaris Hildenborough for hydrogen
production from lactate, formate and ethanol. Among these
substrates, the highest hydrogen production which was about
1489
(320 m3H2 =m3medium ) was acquired from formate as substrate.
While, the hydrogen produced was 21 and 5 m3H2 =m3medium when
lactate and ethanol were used respectively [55].
The MFC-MEC coupled systems
In the MFC-MEC coupled systems, generated electricity in MFC
was used for hydrogen production in MEC with the aim of cost
reduction [9,55,101,132e134]. In some studies which focused
on MFC-MEC coupled systems, the microbial communities
were investigated [101,132,134]. The SEM micrographs which
were taken in these entire studies showed rod-shaped bacte-
ria in both MECs and MFCs biofilms. In addition, the taken
bands of DGGE analysis were similar in MECs and MFCs iso-
lates [101,132,134]. Therefore, there was no significant differ-
ence in microbial communities of both MFC and MEC biofilm
and the population of exoelctrogens in the MFC and MEC
isolates were similar.
16S rDNA analysis in study of Zhang et al. [101] showed
that the acclimatized bacteria consortium was composed of
Bacteroidia, Flavobacteria, Betaproteobacteria, Synergistia, Clos-
tridia and Gammaproteobacteria. The prominent species of this
community are phylogenetically closely related to bio-
ellectrochemically active bacteria [135e138]. However, the
microbial community in this study is different from other
studies results which are primarily Geobacter, Shewanella,
Desulfovibrio and Anaeromyxobacter. This difference was
attributed to the type of inoculum, design and the dependency
of different parts (including hydrogen and electricity produc-
tion parts) to each other in the system design [101].
Conclusions and future prospects
Microbial communities as biocatalyst of bioelectrochemical
processes in MECs have been demonstrated to significantly
affect the overall performance of biohydrogen production. It is
obvious that the control of microbial behaviors have inevi-
table effects in the improvement of MEC efficiency. This re-
view summarizes microbial community analysis methods,
inoculation methods, effective parameters on microbial
communities, biocathodes, the symbiosis of different micro-
bial groups and ways to dominate electrogenesis group.
The future for microbial communities' analysis in MEC, is
associated with several opportunities awaiting further devel-
opment. First, the development of novel, accurate and cost
effective methodologies for in situ detection and quantifica-
tion of microbial communities could facilitate a deeper un-
derstanding of the mechanisms of microbial interactions and
competitions in MEC.
Second, the response to key questions arises from de-
ficiencies and vagueness of previous studies which were
presented in this review including:
 What is the mechanism for initial competition among
various microorganisms during microbial enrichment
process? How can the competition be controlled to form an
effective microbial population in biofilm for enhancement
of biohydrogen production?
1490 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 1 4 7 7 e1 4 9 3
 What are the effective operating parameters which influ-
ence adhesion of microorganisms to electrodes or cohesion
among different species?
 What are the major changes in microbial community of
MEC for biodegradation of complex substrate in different
anode and applied potentials and consequently, does the
applied potential change the rate of sequential reactions of
complex wastewater biodegradation? Besides, what would
be the behavior of suspended species and methanogens
with complex substrates under different applied potentials
and hydrodynamic conditions in the continuous MEC?
 How can the MEC components with their characteristics
such as various substrates with different concentrations,
electrodes characteristics, and membrane selectivity be
controlled to dominate exoelectrogens and/or eliminate
methanogens?
These developments will expedite the commercialization
of MECs as a clean and green technology for biohydrogen
production.
Acknowledgments
This research was supported by Sharif University of Tech-
nology, Vice President for Research Grant G930111.
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