microbial characterization in microbial fuel cells for bioelectricity … · table 1 - sequence and...

1

Microbial characterization in microbial fuel cells for

bioelectricity production

Ana Mafalda Marques Cardoso [email protected]

Instituto Superior Técnico, Lisboa, Portugal November 2014

Abstract The development of new methods combining sustainable energy production and wastewater

treatment are urgently required to solve environmental problems resulting from our fossil fuel

dependence. Microbial fuel cells (MFC) represent a new technology for the production of renewable

energy by direct conversion of organic matter into electricity by bacteria. For MFC optimization, a

complete characterization of the microbial communities that thrive in the anode compartment is

required. The aim of this work is to characterize the microbial populations of two distinct MFC. The

methodology used combined PCR obtaining the 16S rRNA genes, followed by cloning and restriction

profiles analyses after hydrolysis with the restriction enzyme Hae III and sequencing. Databases searches

for sequences homologous to the nucleotide sequences of 16S rRNA encoding genes cloned were

subsequently performed. A total of 960 clones belonging to the Bacteria and Archaea domains,

corresponding to a total of 142 distinct restriction profiles, and 58 different species were obtained. The

estimative of the species relative abundances showed a large number of bacteria belonging to

Burkolderiales, Pseudomonadales, Rhodocyclales e Xanthomonadales, all of them orders of

Proteobacteria. Archaea exhibiting a high relative abundance belonged to the Methanomicrobiales,

order of the Euryarchaeota.

Keywords: Microbial fuel cells, bioelectricity, anaerobic reactors, microbial characterization, PCR

amplification, 16S rRNA gene amplification by PCR, cloning and restriction profiles analyses.

1. Introduction

World population growth is leading to a rapid

increase in urban and industrial waste which

rendering waste management as a significant

environment threat. There’s also a big concern

about the use of fossil fuels because they’re not

renewable and its abusive use will end up with

their extinction. Microbial fuel cells (MFC) are a

new technology in renewable energy that

generates bioelectricity simultaneously with

wastewater treatment. MFCs use

electrochemically active microorganisms as

biocatalysts to oxidize organic matter and

generate electrical current [1]. Besides

carbohydrates, MFCs may work on complex

substrates present in wastewater [2]. A typical

MFC contains two chambers, an anaerobic

anode chamber and an aerobic cathode

chamber separated by a proton exchange

membrane [3]. Electrons produced by bacteria

from the oxidation of substrates move to the

anode and this transfer can occur either by

membrane-associated components, soluble

electron shuttles or nanowires [4]. The

electrons then flow to the cathode through an

external electric circuit which has a resistor

connected, creating bioelectricity (Figure 1).

output of an MFC can be influenced by several

parameters like the anode, fuel cell design or

type of substrates [6]. Depending on the

operational parameters of the MFC, different

metabolic pathways can be used by

microorganisms affecting its performance [3].

Bacteria that are capable of exocellular electron

transfer are called as “exoelectrogens” [4].

Thus, the characterization of the microbial

communities that compose the biofilms in

electrodes is extremely important to achieve a

better understanding of the metabolic

pathways that occur in MFCs and a possible

mailto:[email protected]

2

Figure 2 – Two chambered abiotic cathode MFC R1 (left) and

biocathode MFC R2 (right) set up.

Figure 1 – Schematic diagram of the basic components

in a MFC (adapted from Vinay Sharma and Kundu,

2010) [5]

optimization of the power output [3].In the

latest years, several MFCs were analyzed and it

was confirmed that there is not a typical

bacterial community or a more prominent

organism [7], but they vary from aerobes and

facultative anaerobes towards strict anaerobes

[2]. However, it is known that MFCs using

mixed cultures have some important

advantages over MFCs driven by pure cultures:

more power produced; higher resistance;

higher substrate consumption rates and smaller

substrate specificity [6, 8]. Molecular

techniques based on 16S rRNA sequence

analysis are now widely used to characterize

microbial populations [9].

The aim of this work was to characterize the

microbial diversity in two distinct MFCs based

on the analysis of the 16S rRNA sequences.

2. Materials and Methods

2.1. MFC operation

Samples analyzed in the present work were

from two bioreactors MFCs, reactor 1 (R1) and

reactor 2 (R2), with different operational

conditions (Figure 2). The operation and

maintenance of the reactors were carried out

by Dr. Ramana S. Venkata under the

supervision of Prof. Luís Fonseca (Department

of Bioengineering, DBE, at IST). MFCs anode

chambers and R2 cathode chamber were

inoculated with aerobic mixed consortia from

an industrial wastewater. Both the anode and

cathode chambers were fed with synthetic

wastewater composition comprising sodium

acetate (10 mM) in both anode chambers and

the cathode of R2 was fed with sodium

carbonate (3 mM). R1 cathode chamber was

fed with 50 mM phosphate buffer, as well as

other 50 mM phosphate buffer, nutrient

solution used in the both anode chambers and

cathode chamber of R2. Prior to feeding, pH of

the artificial wastewater was maintained at 7.0

in the anode and cathode chambers. Two

anode chambers were sparged with oxygen

free N2 gas for 20 min to maintain the

anaerobic microenvironment, after the fed

change and sampling. The two MFCs cathode

chambers were continuous supplied with air

through an air-pump to maintain constant the

amount of the electron acceptor. The medium

solution was changed when the voltage

decreased to 50 mV and the suspended

biomass was reserved, forming a complete fed-

batch cycle. MFCs were operated at room

temperature (around 25°C) and electrodes

were connected through a copper wire to a

fixed external resistance of 1000Ω.

After the suspended biomass collection,

samples were immediately frozen at -80°C.

From each compartment from each reactor, it

was collected biomass from two distinct

periods: period in which bioelectricity

production was higher and the period in which

bioelectricity production was lower. This work

presents an analysis of microbial diversity at

both periods.

2.2. Microbial population characterization

The methodology used in this work was

based on [10], and combines sampling, DNA

extraction, polymerase chain reaction (PCR)

amplification, cloning, and sequencing of clones

previously characterized by restriction

fragment length polymorphism analysis (RFLP).

DNA was extracted using the commercial kit

High Pure PCR Template Preparation Kit

3

(Roche) according to the manufacturer’s

instructions. DNA concentration was then

estimated using a spectrophotometer

(NanoDrop ® ND-1000 Technologies) and it’s

quality visually inspected after electrophoresis

in agarose gels (0.8% (w/v)) with TAE buffer

[11]. The PCR reactions were performed using

the primers listed in Table 1.

The amplification mixtures contained in a total

volume of 20 μl, 0.4 μl of each primer, 1.5 μl of

MgCl2, 0.4 μl of DNTPs, 5 μl of template DNA,

0.2 μl of Taqmed DNA polymerase (1 U) and 2

μl of reaction buffer supplied by the

polymerase manufacturer (Citomed).

Amplification products were visualized after

electrophoresis in 0.8% (w/v) agarose gels and

ethidium bromide staining. Amplification

fragments were purified using the kit Gel Pure

(NZYTech). The purified fragments were then

ligated into the TA region of the pCR 2.1 cloning

vector which allows white/blue selection (TA

Cloning Kit, Invitrogen), following the

manufacturer’s instructions (Figure 3). Ligation

mixtures were then used to transform

electrocompetent Escherichia coli DH5-α. The

white clones were selected in LB plates

containing X-gal, supplemented with 50 mg/l of

kanamycin [11]. Plasmid DNA from selected

clones was recovered using the miniprep

procedure [11]. The insertion of DNA fragments

into pCR2.1 was then confirmed by agarose gel

electrophoresis analysis and the DNA

concentration estimated using a

spectrophotometer (NanoDrop ® ND-1000

Technologies).

Plasmidic DNA was digested after adding 1U of

the restriction enzyme HaeIII to recombinant

plasmid preparations. Next, the mixtures were

subjected to electrophoresis in 30%

polyacrylamide gels in TBE (10x) (TBE 10x: 108

g/L Tris Base, boric acid 55 g/L, 7.44g

Na2EDTA•2H2O), deionized water, ammonium

persulfate (APS) (10%) and

tetramethylethylenediamine (TEMED). After the

polymerization, the gel was subjected to

electrophoresis running at 110V and then

visualized in a UV system (BioRad, Universal

Hood II). Finally, the restriction patterns

obtained were compared to pool them together

concerning their profile, migration distance and

relative intensity. Unique profiles were assumed

to belong to different clones. DNA sequences

were analyzed with the VecScreen tool from

National Center for Biotechnology Information

(NCBI) to eliminate DNA sequences of the pCR

2.1 cloning vector. The sequences obtained

were aligned using in the Basic Local Alignment

Search Tool (BLAST) resulting in the

identification of the organisms with sequences

homologous to the sequences obtained.

Alignment of the sequences identified in this

work with sequences retrieved from databases

was performed using the CLUSTAL W software.

3. Results

3.1 Sample identification

To easy the identification of samples

obtained after the PCR reactions, the

nomenclature summarized in Table 2 was

adopted to label sequences which indicates

the reactors (1 and 2), the chambers (C and A),

the bioelectricity phase production and primers

used (A for Archaea or B for bacteria).

Domain Annealing tempera-ture (°C)

Forward primer (5’-3’)

Reverse primer (3’-5’)

Expected amplicon size (bp)

Bac

teri

a

70

CCAGATCCTACGGGAGGC

AGC

CTTGCTCGGGCCCCGTCA

ATTC

605 [12]

Arc

hae

a

56 ACTGCTCAGTAAC

ACGT

CTCCCCCGCCAATTCTTTA

803 [13]

Table 1 - Sequence and relevant characteristics of the

oligonucleotide primers designed and used in this work

Figure 3 – Schematization of the principle of cloning in the

PCR2.1 vector (TA Cloning Kit, Invitrogen).

4

3.2 Gel electrophoresis analysis

After the PCR reactions, the existence and

integrity of the purified genomic DNA were

confirmed by gel electrophoresis (data not shown).

The absence of bands in R1A for the lower

production phase of bioelectricity, when using

Archaea primers in PCR reactions, suggested that

there were no Archaea in the anodic chamber.

There were no Archaea bands in R2A and R2C for

the lower production phase of bioelectricity, also

suggesting the absence of Archaea in both

chambers. For the higher production phase of

bioelectricity it wasn’t possible to obtain any

genomic DNA. In this case, we considered the

absence of Archaea in the cathode chamber of R2.

Using bacteria primers, the results showed great

band intensity for both reactors (anode and

cathode) in the two phases of bioelectricity

production. These results were confirmed and were

in conformity with the spectrophotometer results.

3.3. RFLP analysis of clones

After amplification reactions and purification of the

amplimers using a commercial kit, were cloned into

the pCR2.1 cloning vector. A total of 960

recombinant plasmids were obtained from the DNA

extracted from both reactors (anode and cathode)

in the two phases of bioelectricity production. The

RFLPs of the recombinant plasmids was analyzed

with HaeIII. The comparison of the restriction

profiles of the clones allowed their grouping into

distinct patterns. Table 3 summarizes the number of

clones obtained in each assay by the number of

different profiles obtained.

Total number of

clones obtained

Effective number

of different

profiles

R2A_B1 96 8

R2A_A1 119 9

R2C_B1 120 6

R1A_B1 87 8

R1C_B1 126 8

R1A_A1 68 7

R2C_B2 135 20

R2A_B2 41 7

R1C_A2 72 0

R1C_B2 48 8

R1A_B2 48 4

Total 960 85

After sequencing of the 142 initial profiles,

only 85 corresponded to different clones. The

following figures illustrate examples of

restriction profiles of the clones obtained for

the two reactors, using primers for the 16S

rRNA of bacteria (Figure 7) or Archaea (Figure

8).

Higher production

phase of bioelectricity

Lower production


Anode Cathode Anode Cathode

R 1

Bacteria R1A_B1* R1C_B1 R1A_B2 R1C_B2

Archaea R1A_A1* R1C_A1 R1A_A2 R1C_A2

R 2

Bacteria R2A_B1 R2C_B1 R2A_B2 R2C_B2

Archaea R2A_A1 R2C_A1 R2A_A2 R2C_A2

Figure 7 - RFLP profiles of the distinct clones of bacteria

identified in this work and obtained after restriction with

HaeIII and electrophoresis. (A)-Burkholderiales; (B)-

Rhodocyclales; (C)-Xanthomonadales; (D)-Rhizobiales; (E)-

Nitrosomonadales; (F)-Enterobacteriales; (G)-

Desulfuromonadales; (H)-Sphingomonadales; (I)-

Pseudomonadales; (J)-Caulobacterales; (K)-Flavobacteriales;

(L)-Sphingobacteriales.

Figure7 - RFLP profiles of the distinct clones of bacteria


HaeIII and electrophoresis. (A)-Burkholderiales; (B)-

Rhodocyclales; (C)-Xanthomonadales; (D)-Rhizobiales; (E)-

Nitrosomonadales; (F)-Enterobacteriales; (G)-

Desulfuromonadales; (H)-Sphingomonadales; (I)-

Pseudomonadales; (J)-Caulobacterales; (K)-Flavobacteriales;

(L)-Sphingobacteriales.

Figure 8 - RFLP profiles of the distinct clones of archaea


HaeIII and electrophoresis. (A)-Methanosarcinales; (B)-

Methanobacteriales; (C)-Methanomicrobiales; (D)-

Methanomassiliicoccus; (E)-Nitrososphaerales.

Figure 8 - RFLP profiles of the distinct clones of archaea


Table 2 – Adopted nomenclature to label the reactors, the

chamber, bioelectricity phase production and primers used (for

Archaea or bacteria). The samples marked with an asterisk were

analyzed by Andreia Fernandes during an internship under the

supervision of professors Luís Fonseca and Jorge Leitão (IST) and

have been included for a better comprehension of the work.

Higher production


Lower production


Anode Cathode Anode Cathode

R 1

Bacteria R1A_B1* R1C_B1 R1A_B2 R1C_B2

Archaea R1A_A1* R1C_A1 R1A_A2 R1C_A2

R 2

Bacteria R2A_B1 R2C_B1 R2A_B2 R2C_B2

Archaea R2A_A1 R2C_A1 R2A_A2 R2C_A2

Table 2 – Adopted nomenclature to label the reactors, the

chamber, bioelectricity phase production and primers used (for

Archaea or bacteria). The samples marked with an asterisk were

analyzed by Andreia Fernandes during an internship under the

supervision of professors Luís Fonseca and Jorge Leitão (IST) and

have been included for a better comprehension of the work.

Table 3 – Comparison between the total numbers of clones

obtained and the effective number of different profiles.

Table 3 – Comparison between the total numbers of clones

obtained and the effective number of different profiles.

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3.4. Microbial population composition

After sequencing, the nucleotide sequences

corresponding to partial 16S rRNA encoding genes

were used to perform database searches for

homologs. For each sequence obtained, the

organism which 16S rRNA encoding gene

sequence was closer to our sequence was

obtained. Based on these results, filogenetic trees

were obtained (data not shown). Tables 4 to 11

summarize the species identified in both phases

of bioelectricity production of the two reactors in

the anode and cathode chamber. For each

sequence obtained is shown the closest specie as

well as the max score, total score, query cover, e-

value and identity.

Table 4 – Microbial species identified with sequences

homologous to the sequences obtained by PCR in the anodic

chamber of R1 in the higher production phase of bioelectricity.




For each sequence obtained the closest species, as well as the

max score, total score, query cover, e-value and identity are

shown.



chamber of R1 in the lower production phase of bioelectricity.






shown.


homologous to the sequences obtained by PCR in the

cathodic chamber of R1 in the higher production phase of

bioelectricity.


homologous to the sequences obtained by PCR in the

cathodic chamber of R1 in the higher production phase of

bioelectricity. For each sequence obtained the closest

species, as well as the max score, total score, query cover, e-

value and identity are shown.

Table 7 – Microbial species identified with sequences homologous

to the sequences obtained by PCR in the cathodic chamber of R1

in the lower production phase of bioelectricity.


to the sequences obtained by PCR in the cathodic chamber of R1

in the lower production phase of bioelectricity. For each sequence

obtained the closest species, as well as the max score, total score,

query cover, e-value and identity are shown.


to the sequences obtained by PCR in the anodic chamber of R2 in

the higher production phase of bioelectricity.


to the sequences obtained by PCR in the anodic chamber of R2 in

the higher production phase of bioelectricity. For each sequence

obtained the closest species, as well as the max score, total score,

query cover, e-value and identity are shown.






chamber of R2 in the lower production phase of bioelectricity. For

each sequence obtained the closest species, as well as the max

score, total score, query cover, e-value and identity are shown.


homologous to the sequences obtained by PCR in the cathodic




chamber of R2 in the higher production phase of bioelectricity. For

each sequence obtained the closest species, as well as the max

score, total score, query cover, e-value and identity are shown.

6

Considering the number of clones which correspond to each restriction profile, an estimative of the relative abundance of the different organisms in both reactors for the two phases of bioelectricity production is shown (Figures 9 to 12). However, it should be noted that there results can have some errors associated due to possible bias introduced by the PCR reactions. Nevertheless, the following figures compare the relative abundance of microorganisms in both phases of bioelectricity production.

Figure 9 – Relative abundance comparison of the identified

species in anodic chamber of R1 in higher or lower

production phase of bioelectricity.

Figure 10 – Relative abundance comparison of the

identified species in cathodic chamber of R1 in higher or

lower production phase of bioelectricity.


identified species in anodic chamber of R2 in higher or



identified species in cathodic chamber of R2 in higher or


Based on the clone library obtained and in the

relative abundance of each clone analyzed by

the RFLP method, we found that Proteobacteria

is the most abundant bacterial phylum,

representing more than 95% of the clones.

Regarding to the order level, Pseudomonadales

and Burkholderiales represent the most

abundant organisms corresponding to 33% and

23%, respectively, of the total Proteobacterias.

On the other hand, the strictly carbon dioxide

reducing Methanomicrobiales, Euryarchaeota

phylum, is the most abundant order,

corresponding to 90% of total archaeal clones.

At the end, we have identified 45 different

species of bacteria and 13 species of Archaea.

3.5. Discussion

3.5.1. Reactor 1

In the anodic chamber of reactor 1, 87

clones for bacteria and 68 clones for Archaea









shown.

7

were obtained for the higher production phase

of bioelectricity. In the lower production phase

of bioelectricity 48 clones of bacteria in the

anode chamber (R1A_B2) were obtained.

However, in the cathodic chamber (R1C_B1)

126 bacterial clones were obtained for the

higher prodution phase of bioelectricity, 72

archaeal clones and 48 bacterial clones for the

lower prodution phase of bioelectricity. Figure

11 shows that in R1 on higher production phase

of bioelectricity, Pseudomanadales and

Rhodocyclales were the bacterial orders in

anode chamber. However, these classes were

absent in samples from the lower production.

Instead, sequences similar to that of members

of the Burkholderiales e Rhodospirillales were

retrieved. These results suggest that although

Burkholderiales and Rhodospirillales are

commonly identified in MFCs, their role may

not have such great importance in bioelectricity

production. Their role also may be dependent

of other species such Pseudomonadales.

A large percentage of the sequences

identified in the cathodic chamber of R1, in the

higher production phase of bioelectricity, were

similar to Burkholderiales, Pseudomonadales

and Enterobacteriales orders (Figure 12).

However, in the lower production phase of

bioelectricity, a clear decrease of

Burkholderiales and a total disappearance of

Enterobacteriales was registered. Sequences

belonging to Pseudomonadales orders were

also identified in this phase representing the

most abundant order. In this phase,

Pseudomonadales, Xanthomonadales and

Rhizobiales were the most represented orders

in the cathode chamber.

R1 was characterized by a great abundance

of β-Proteobacteria, especially of species of the

Burkholderiales order, which were indentified

in both the chambers in both production

phases of bioelectricity. For example, in the

cathodic chamber and for the higher

production phase of bioelectricity (R1C_B1),

sequences belonging to the Burkholderiales

order represented about 73% of the total 126

clones obtained. In the anodic chamber, for the

same phase of bioelectricity production

(R1A_B1), Burkholderiales represented only 1%

of the total clones and 3% and 11%,

respectively, in the cathodic (R1C_B2) and

anodic (R1A_B2) chambers in the lowest

production phase of bioelectricity. Species from

the Diaphorobacter and Acidovorax genera

(Burkholderiales order), have already been

found in MFCs enriched with domestic

wastewater [14]. Acidovorax could be related

with the production of bioelectricity [15].

The Rhodocyclaceae comprises gram-

negative bacteria that belong to the β-

Proteobacteria. They only weren’t identified in

the cathodic chamber in the lowest production

phase of bioelectricity (R1C_B2).

Rhodocyclaceae represented about 9% of

clones obtained in the higher production phase

of bioelectricity in the cathode (R1C_B1), 30%

of clones obtained in the higher production

phase of bioelectricity in the anode (R1A_B1)

and 78% of clones obtained in the lower

production phase of bioelectricity in the anode

(R1A_B2). Members of this family are sulfate

and chlorate reducing bacteria, iron reducing

bacteria and are characterized by their high

tolerance to oxygen [16].

Pseudomonas is one of the genera more

represented in reactor 1 and have been used to

supply the MFC’s anodes in order to optimize

the bioelectricity production [17]. Rabaey et al.

(2004) [8] evaluated the electrochemical

potential of a pure culture of Pseudomonas to

perform the electronic transfer directly into the

anode. In the present work, 24% of the clones

obtained in the higher production phase of

bioelectricity (R1C_B1) belonged to the

Pseudomonas genus, which represents 60% of

clones obtained in the higher production phase

of bioelectricity in anode (R1A_B1) and 50% of

clones obtained in the lowest production phase

of bioelectricity in cathode (R1C_B2).

Pseudomonas and Burkholderia are two aerobic

genera which can use oxygen or another

electron acceptor. For that reason, they can use

oxygen or the anode electrode as electron

acceptors, which can explain their high relative

abundance on both chambers of reactor.

Species of Enterobacteriales, like Serratia or

Shigella boydii were identified in this reactor.

Enterobacteriales represented about 32% of

the 126 clones obtained from the cathode in

the higher production phase.

8

The Bacterioidetes phylum was also

identified in reactor 1. Organisms like

Flavobacteriales (Chryseobacterium) and

Cytophagales (Leadbetterella byssophila) were

identified, being this phylum one of the most

abundant in the cathode chamber [18].

Bacterioidetes and Proteobacterias are essential

to oxygen and nitrate reduction in the cathode

[19] and they represented about 2% and 6% of

the clones obtained, respectively, in the higher

production phase of bioelectricity. In the higher

production phase of bioelectricity of the anode

(R1A_B1) a sequence with homology to

Magnetospira, belonging to Rhodospirillales

which are photosynthetic bacteria was obtained.

However their importance is still unknown.

Rhizobiales were identified in all compartments

with the exception of the lower production

phase of bioelectricity in the anode and

represented about 9% of clones obtained in the

higher production phase of bioelectricity in the

cathode (R1C_B1) and 18% in the lower

production phase of bioelectricity (R1C_B2). The

Nitrobacter, Mesorhizorbium and

Methylobacterium organo were members of the

Rhizobiales identified and that are recognized as

electrochemical active aerobic bacteria [20]

Archaea were identified in the higher

production phase of bioelectricity anode

(R1A_A1) and in the lower production phase of

bioelectricity cathode (R1A_A2). In the first case,

Methanosphaerula palustris and Methanolinea

species of the Methanomicrobiales order were

identified. Methanosaeta concilii, belonging to

the Methanosarcinales order, is characterized by

acetate consumption and methane production.

In the cathode and for the lower production

phase of bioelectricity (R1C_A2), species

Methanosaeta concilii, Methanolinea and

Methanosaeta were identified. Although

Archaea are rarely studied in MFCs, it is very

common to find them, especially methanogenic

species [21]. It is also common to find them in

anode where electrons can be used to reduce

carbon dioxide to methane [22]. At the cathode,

the existence of Archaea is greatly reduced due

to exposure to O2 and the organic substrate

competition by facultative anaerobes [23].

3.5.2. Reactor 2

In the anodic chamber of R2 (Figure 11) a total

of 96 clones of bacteria and 119 clones of

Archaea were obtained for the higher

production phase of bioelectricity. In the lower

production phase of bioelectricity, 41 bacterial

clones were obtained for the anode. In cathodic

chamber, 120 bacterial clones in the higher

production phase of bioelectricity and 135

bacterial clones in the lowest production phase

were obtained. Sequences with homology to

the Pseudomonadales order presented the

higher abundance in the higher production

phase of bioelectricity followed by the

Rhodocyclales order. However, in the lowest

production phase of bioelectricity, organisms of

Pseudomonadales order could not be detected.

On the other hand, the relative abundance of

Burkholderiales, Rhodocyclales and

Desulfuromonadales, was higher. These results

suggest that the Pseudomonadales

microorganisms are those that contributed

most significantly to the production of

bioelectricity in the anode.

In the cathodic chamber (Figure 12) a

great relative abundance of organisms

belonging to the Burkholderiales and

Xanthomonadales orders in the higher

production phase of bioelectricity were

obtained. In the lower phase production, a

higher microbial diversity was detected being

Xanthomonadales the most represented order.

In the higher production phase of bioelectricity

of anode, a high relative abundance of

Pseudomonas was detected, representing

about 78% of the 96 clones identified.

Geobacter was identified with a relative

abundance of about 3% in the higher

production phase and 13 % in the lower

production phase. Geobacter species are

considered as highly efficient in the electron

transfer to the electrode, capable of

transferring most of electrons obtained from

the consumption of acetate directly to the

electrode [24]. In the lower production phase

of bioelectricity, Propioniovibrio militaris,

belonging to the order of Rhodocyclacles, was

the most abundant specie at the anode,

representing 56% of total clones. Regarding the

9

cathode, for the higher energy phase

production (R2C_B1), only organisms belonging

to the phylum Proteobacteria were identified,

60% belonging to the order of Burkholderiales.

From the 120 clones obtained, 29% belonging

to the order Xanthomonadales, represented by

Dokdonellas and Rhodanobacter, genera that

have been found in several anaerobic reactors

[25]. The lowest production phase of

bioelectricity cathode (R1C_B2) gave the

highest biodiversity. Species of the phylum

Proteobacteria, Firmicutes, Bacteroidetes,

Chloroflexi, and Acidobacteria were found.

Homologous sequences to the genus

Stenotrophomonas, of the Xhantomonadales

order, were the most abundant, representing

about 28% of the total.

From the Archaea domain, only

homologous sequences were obtained from

the anode during the highest production phase

of bioelectricity (R2A_A1). Most of the species

were of the genus Methanolinea, belonging to

the order of Methanomicrobiales, with a 82%

relative abundance (Figure 11). The

methanogens belonged to the phylum

Euryarchaeota. A Candidatus Nitrososphaera

gargensis, belonging to Nitrososphaerales order

from the phylum Thaumarchaeota was also

found.

4. Conclusions In this work, samples from two

separate reactors MFC supplied with waste

water, for the production of bioelectricity were

analyzed in order to gain clues on the microbial

populations. From a total of 960 clones

obtained, 142 unique profiles could be

distinguished 58 different species between

bacteria and Archaea. Sequences with a high

similarity to those from Burkholderiales,

Pseudomonadales, Rhodocyclales and

Xanthomonadales, were the most abundant, all

belonging to the phylum of Proteobacteria.

Among the Burkholderiales were identified 8

different species, were Piscinibacter is the most

abundant. Pseudomonas aeruginosa was the

only species of the Pseudomonadales.

Rhodocyclales were represented by 4 different

species, were Rhodocyclaceae was the most

abundant and finally from the order of

Xanthomonadales, where 3 different species

were identified, Rhodanobacter was the most

abundant. Methanolinea, belonging to the

order Methanomicrobiales and the

Euryarchaeota phylum, were the more

represented Archaea in this work.

The high diversity of species, including

various phyla probably reflects the initial

inocula use, sludge from a domestic

wastewater treatment plant. It also might

reflect that MFCs undergo a process of "self-

optimization" to a better functional

performance of the system. This process should

make a selection of species, depending on the

interactions between them, the amount and

type of substrates available, etc. In a long-term

perspective, it is important to know such

processes, which demonstrates the usefulness

of MFCs systems for the enrichment of

populations functionally stable, capable of

degrading organic compounds while making the

transfer of electrons extracellularly.

Although there are no identical microbial

communities in MFCs, our results contribute for

the practical application of MFC systems in

wastewater treatment. The success of an MFC

for wastewater treatment depends on the

concentration and the degradability of the

organic matter, temperature and absence of

toxic substances and on the microbial

communities’ composition and performance

[6]. Although highly effective large-scale MFCs

are still out of our reach, this is a promising

technology in which researchers and engineers

are struggling to overcome several obstacles

still underlying. The increasing pressure on the

environment and renewable energy will

certainly contribute to the development of this

technology.

5. References

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