Advances in Bioscience and Biotechnology, 2016, 7, 329-335

Published Online July 2016 in SciRes. http://www.scirp.org/journal/abb

http://dx.doi.org/10.4236/abb.2016.77031

Generation of Electricity by Electrogenic

Bacteria in a Microbial Fuel Cell Powered
by Waste Water
Zakira Naureen*, Zainab Ali Rashid Al Matani, Miyassa Nasser Al Jabri,
Saif Khalfan Al Housni, Syed Abdullah Gilani, Fazal Mabood, Saima Farooq,
Javid Hussain, Ahmed Al Harrasi
Department of Biological Science and Chemistry, University of Nizwa, Nizwa, Oman

Received 27 September 2016; accepted 4 July 2016; published 7 July 2016

Copyright © 2016 by authors and Scientific Research Publishing Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/

Abstract
The present study aimed at isolation characterization and evaluation of electrogenic bacteria for
electricity generation using waste water. In this context, waste water samples were collected from
University of Nizwa waste water treatment plant. A total of eight distinct bacterial isolates were
isolated from these samples by serial dilution and plating on LB Agar medium. The bacterial iso-
lates were than grown at different temperatures and pH. DNA from bacterial samples was isolated
and 16S rRNA gene amplification was carried out. The 16S rRNA gene PCR products were directly
sequenced and the resulting sequence was blasted using BLASTn. Based on BLAST results, the
bacterial strains were identified. The bacteria were used in different combinations to generate
electricity from waste water in microbial fuel cells constructed using plastic bottles. The microbial
isolates were found to produce varying levels of currents and their electrogenic potential in waste
water was observed to increase with the passage of time.

Keywords
Electricity Production, Electrogenic Bacteria, Waste Water, Microbial Fuel Cell

1. Introduction
The world energy demand and the corresponding need to find energy sources alternative to fossil fuels are on an
ongoing increase. Microbial Fuel Cell (MFC technology) powered by wastewater is one such effort to provide
*
Corresponding author.

How to cite this paper: Naureen, Z., Al Matani, Z.A.R., Al Jabri, M.N., Al Housni, S.K., Gilani, S.A., Mabood, F., Farooq, S.,
Hussain, J. and Al Harrasi, A. (2016) Generation of Electricity by Electrogenic Bacteria in a Microbial Fuel Cell Powered by
Waste Water. Advances in Bioscience and Biotechnology, 7, 329-335. http://dx.doi.org/10.4236/abb.2016.77031
Z. Naureen et al.

an alternative inexpensive and eco-friendly energy source [1] [2]. Besides generating energy, MFCs offer a great
potential to utilize chemical energy in wastewater by converting to electrical energy through respiration of mi-
crobial inhabitants of wastewater [2]. Wastewater is an energy rich source for growth of several anaerobic and
facultative bacterial species which have the capability to transfer electrons to an anode, as a terminal electron
acceptor and thus are classified as electrogenic bacteria (EB; [3]-[6]). These EB have been documented to gen-
erate electricity by assimilating a variety of sources [7]-[10] like carbohydrates [11], textile effluents [12], waste
matter in land fill [1], sea sediments and waste water [1]; however a very few reports are available where waste
water is used as fuel for electrogenic bacteria which utilize organic and inorganic compounds in it as fuel to
generate electricity. The present study will focus on electrogenic potential of facultative anaerobic bacteria to
generate the electricity in a fabricated two-chambered MFC powered by waste water.

2. Material and Methods

2.1. Collection of Samples
The waste water samples were collected in sterile containers from University of Nizwa. The samples were stored
at 4˚C for further studies.

2.2. Isolation of Bacteria from Waste Water Sediments

The waste water samples were allowed to stand for 24 hours and the sediments were collected and serially di-
luted with saline water (0.9% w/v NaCl) up to 10−5 dilutions. From each dilution 0.1 mL was then spread on the
LB agar plate (Tryptone 10.0 g, NaCl 10.0 g, Agar 20.0 g and Yeast extract 5.0 g in 1000 mL distilled water)
and incubated for 24 h at 30˚C. Morphologically distinct bacterial colonies were purified and further studied for
their Gram staining properties [13].

2.3. Physiochemical Growth Optimization of Bacteria

The morphologically distinct colonies were further inoculated in LB broth and kept at different pH [13] [14] and
different temperature (27˚C, 37˚C, 47˚C and 57˚C) in an orbital shaker to study the growth pattern of these bac-
teria by measuring absorption at 660 nm, against the sterile LB broth as blank [14] using an ELISA reader.
These bacterial isolates were further inoculated in waste water samples and then their growth parameters were
recorded and compared with that in LB broth. The best surviving bacterial strains were used further in MFCs.

2.4. Electricity Generation and Identification of Potent Electrogenic Bacteria

After optimizing growth and MFC parameters, the isolates were studied for their electrogenicity within 500 mL
LB broth culture (24 h old) as anolyte against 500 mL waste water solution as catholyte. Electrogenicity were
recorded in terms of OCV. The potentially active electrogenic bacteria were identified using 16S rRNA gene
cloning, sequencing and analysis using MEGA blast [15].

2.5. MFC Fabrication and Operation

Two polycarbonate bottles (500 mL) were used to construct an H-shaped MFCs. The bottles were linked with
PVC pipes of various lengths such as (5 cm × 1 cm; 10 cm × 2 cm etc.) for preparing a salt bridge. The salt
bridges were filled by boiled cooled sodium chloride (15%) solution containing 5% agar. The salt bridges were
fixed to the bottles with the aid of epoxy adhesive. The waste water cultures of selected bacterial strains were
used as anolyte without any pretreatment and various concentrations of waste water were used as a catholyte in
the MFC setups. Plain graphite plates (5 - 5 cm; 10 mm thick; surface area, 70 cm2) were used as electrodes.
Circuit connections were set with the copper wires fixed into the drilled holes of the electrodes and sealed with
epoxy resin to avoid corrosion of copper wire [12].
The MFCs were sterilized by thorough rinsing with Ethanol (70% v/v) and UV irradiated for 30 min. The ste-
rilized MFC chambers were filled with respective electrolytes and kept at room temperature 28˚C ± 2˚C for one
day. Electricity generation was recorded using a multidigital meter (UNI-DT830D, Uni-Trend Group Ltd.,
Kowloon, Hong Kong) after 1, 7 and 21 days.

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3. Results
Waste water samples were collected from UoN waste water treatment plant and allowed to stand for 24 hrs. The
sediments were collected and diluted and directly plated on respective media for isolation of inhabiting bacteria.
The morphologically distinct colonies were selected for further studies.

3.1. Determination of Phenotypic Characteristics

Gram Staining
Out of 8 distinct bacterial isolates screened 7 were Gram negative and one was Gram positive. All bacterial
strains were catalase positive and facultative anaerobes (Table 1).

3.2. Bacterial Growth Optimization at Different Temperatures

The isolates were grown for 7 days at 27˚C, 30˚C, 37˚C, and 55˚C. We observe that most of isolates were able to
grow at 27˚C and the other isolates were able to grow best at 37˚C, and weak observations of growth were taken
at 30˚C and 55˚C. Four reading were taken with three replications for each isolates to calculate the number of
cells as shown in Table 2.

3.3. Bacterial Growth Optimization at Different pH

The isolates were grown for 7 days at pH 4, 5, 9 and 11 respectively. We observe that most of isolates were able
to grow at pH 5 while other isolates were able to grow best at pH 9. Weak o growth of bacteria was observed at
pH 4 and pH 11. For each isolate four readings were recorded with three replications to calculate the number of
cells as shown in Table 3.

3.4. Identification of Isolates by 16S rRNA

16S rRNA was performed for all isolates. The forward primer was complementary to the 5’-end of 16S rRNA
and the reverse primer was complementary to 3’- of the 16S rRNA (Young et al., 1991). The amplified PCR
products were cleaned and directly sequenced. The obtained sequence was trimmed, chimera removed and
blasted on BLAST n of NCBI. The bacterial species were identified on the basis of maximum similarity with
known genera and then the sequences were submitted to Genbank and accession numbers were obtained.

Table 1. Physiological characteristics and 16S rRNA gene sequencing based identification of bacterial isolates obtained from
waste water samples.

Sequences Strain ID Identification Gram staining Respiration Accession numbers

Facultative
seq1 Z1 Chromobacterium sp. 1 Gram negative KT347176
anaerobic

Facultative
seq2 Z2 Amantichitinum ursilacus Gram negative KT347177
anaerobic

Facultative
seq3 Z3 Chromobacterium sp Gram negative KT347178
anaerobic

Facultative
seq4 Z4 Bacillus licheniformis Gram positive KT347179
anaerobic

Facultative
seq5 Z5 Enterobacter sp Gram negative KT347180
anaerobic

Facultative
seq6 Z6 Unidentified bacteria Gram negative NOT IDENTIFIED
anaerobic

Facultative
seq7 Z7 Escherichia coli Gram negative KT347181
anaerobic

Facultative
seq8 Z8 Citrobacter sp. Gram negative KT347182
anaerobic

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Table 2. Bacterial growth at different temperatures.

Bacterial isolates

Temperature Chromobacterium Amantichitinum ursilacus Chromobacterium Bacillus Enterobacter Unidentified Escherichia Citrobacter
licheniformis
sp. 1 Z1 Z2 sp. Z3 sp. Z5 bacteria Z6 coli Z7 sp. Z8
Z4

27˚C 1.53 × 109 1.25 × 109 1.07 × 109 1.55 × 109 3.0 × 109 3.26 × 108 2.96 × 109 1.49 × 109
30˚C 1.36 × 109 7.26 × 108 1.28 × 109 1.57 × 109 6.24 × 108 7.34 × 108 7.28 × 108 1.43 × 109
37˚C 1.45 × 109 5.3 × 108 1.34 × 109 1.42 × 109 2.98 × 108 3.52 × 108 3.99 × 108 1.6 × 109
55˚C 6.32 × 108 2.25 × 108 6.02 × 108 6.01 × 108 2.55 × 108 2.57 × 108 2.64 × 108 1.11 × 109

Table 3. Bacterial growth at different pH.

pH Bacteria isolates

Bacillus
Chromobacterium Amantichitinum ursilacus Chromobacterium Enterobacter Unidentified Escherichia Citrobacter sp.
licheniformis
sp. 1 Z1 Z2 sp. Z3 sp. Z5 bacteria Z6 coli Z7 Z8
Z4

4 1.20 × 109 1.03 × 108 5.37 × 108 1.03 × 108 1.51 × 108 3.26 × 108 1.32 × 108 1.31 × 108

5 3.75 × 108 1.26 × 109 1.17 × 108 7.08 × 108 9.32 × 108 1.31 × 108 2.87 × 108 5.35 × 108

9 5.37 × 108 7.34 × 108 1.03 × 108 1.65 × 109 5.35 × 108 3.52 × 108 1.60 × 109 1.6 × 109
11 1.11 × 108 3.75 × 108 1.43 × 108 1.36 × 109 1.88 × 108 2.57 × 108 3.35 × 108 6.47 × 108

3.5. Sequences Analyses and Phylogenic Study

The data was set, aligned, curetted, and phylogeny program shown (Figure 1). Sequence analysis through
BLAST search identified seven of the total eight bacterial strains as Chromobacterium sp. (two strains Z1 and
Z3), Amantichitinum ursilacus (Z2), Bacillus licheniformis (Z4), Citrobacter (Z8), Escherichia coli (Z7), and
Enterobacter sp. (Z5) while one of the bacterial strains remained unidentified (Z6).
As the phylogenetic tree shows, the isolates were divided into two major groups and five subgroups, the
members of one of the subgroups (Chromobacterium sp.) were 100% related to each other. Both the Chromo-
bacterium sp. Z1 & Z3 were grouped together because they had higher sequence similarity. However, Amanti-
chitinum ursilacus Z2 and Bacillus licheniformis also sub-clustered with Chromobacterium sp. The Citrobacter
sp. Z8 was separated from rest of all the bacterial strains showing that it has a low conservative sequence. In a
second sub-cluster, Enterobacter sp. Z5, Escherichia coli Z7 and unidentified bacteria were grouped together. In
the second sub-cluster, unidentified bacterium was separated from the group which shows that its 16S rRNA
sequence had more nucleotide variations.
For constructing the tree FASTA sequences were first aligned using Clustal Omega and then submitted to
MEGA 5 (Bootstrap number 1000).

3.6. MFC Fabrication and Operation

Microbial fuel cells were operated with different anode and cathode compositions and with different salt bridges.
The best results were obtained for NaCl salt bridge of 10 cm length. Different bacterial isolates showed varying
level of electricity generation when tested against varying concentration of waste water. The best results were
obtained for 50% wastewater concentration. Highest electric current at 0 days was observed for bacterial isolate
Enterobacter sp. Z5 (Table 4). The Microbial fuel cells were than left at room temperature (30˚C) for 7 days af-
ter which OCV were recorded again. An increase in OCV was observed for all bacterial isolates with Chromo-
bacterium sp.1 Z1, Amantichitinum ursilacus Z2 and Enterobacter sp. Z5 showing high levels of electric cur-
rents. The bacterial isolates in the respective fuel cells were further allowed to respire in the MFCs for 21 days.
Tremendous increase in electricity generation was observed for all bacterial strains with highest being observed
for Amantichitinum ursilacus Z2.

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Figure 1. Phylogenetic analysis of bacterial isolates.

Table 4. Electricity production by bacterial isolates at different days.

Electricity production
Bacteria isolates
First trial (mV) 1 days Second trial (mV) 7 days Third trial (mV) 21 days
Chromobacterium sp. 1 (Z1) 86 141 430
Amantichitinum ursilacus (Z2) 37 143 645
Chromobacterium sp. (Z3) 63 66 230
Bacillus licheniformis (Z4) 10 16 222
Enterobacter sp. (Z5) 114 135 420
Unidentified bacteria (Z6) 67 72 Not detected
Escherichia coli (Z7) 29 78 625
Citrobacter sp. (Z8) 16 17 460

4. Discussion
The present study states that microbial inhabitant of waste water have electrogenic potential and they can ma-
nifest this potential if they are properly re-inoculated into their habitat. This leads to the production of bioelec-
tricity in the fabricated MFCs. A total of 8 distinct bacterial isolates from waste water sediments were isolated
and their 16S rRNA gene sequence analysis showed that these belong to different genera. Bacterial isolate 6 did
not show specific resemblance to any known genera suggesting that it might be novel specie/genera. This will be
further studied by whole genome sequence analysis coupled with biochemical identification methods. Microbial
fuel cells constructed by using 1000 ml PVC bottles and connected with 15 cm long and 4 cm diameter salt
bridge were used to generate electricity by microbial cell suspension cultures in various concentrations of waste
water. Microbial cultures served as anodes while waste water concentrations served as cathodes. This analysis of
different salts for the Salt Bridge recommended the use of 10% NaCl concentration as optimized salt in the salt
bridge to facilitate easy ion flux. The sodium chloride in the salt bridge yielded good OCV might be because of
its good electrolytic property [16]. While 15 cm length of salt bridge proved better with 4 cm diameter might be
because of less density of agar in longer length and easy flow ions through wider diameter. This result is sup-
ported by similar observations by other researchers [16]. It was reported that most of the exogenous mediators
are toxic for the microbes [2]. Electricity generation from the 100% wastewater could not produce good yield
might be effect of ions present in both the electrolytes. An increase in the OCV was observed for all bacterial
isolates after 7 and 21 days of incubation suggesting that high flow of electrons through salt bridge with in-
creasing time period. The OCV of 645 mV was generated by the isolate Amantichitinum ursilacus (Z2) was
nearer to that of maximum OCV of 800 mV [16] reported until now. This implies that the bacterial isolate
Amantichitinum ursilacus (Z2) might have electrogenic properties like C-type cytochromes [17] or conductive

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nano-wires [18] on its cell membrane to generate electric potential [19].

The electrogenic potential of these bacteria may be attributed to their ability to adapt to different pH and dif-
ferent temperatures. For instance most of the bacterial isolates grew well at 30˚C and this might be the reason
for increase in electrogenic potential of these bacteria when they were left in MFC at a room temperature of
30˚C. It has been previously demonstrated that the power density of MFCs is seriously affected by operation
temperatures. For instance the conductivity was reported to increase by 2% ˚C−1 which could be attributed to in-
crease in the activity of anode microorganisms, distribution proportion of nutrients, growth of electrochemically
active microbes, conductivity of electrolyte and chemical reaction rate [20]. Therefore, an increase in tempera-
ture results in decrease in the internal resistance of MFC thus minimizing the energy loss and resulting in an in-
crease in electrogenic potential of the system [21]. Moreover different bacterial isolates had different electro-
genic processes, some strains were capable of producing electricity rapidly (Z5) while others enriched slowly
and showed an increase in electricity generation after 7 & 21 days (Z2 & Z7). It was probably because Z5 had
fast growth rate and adhered to electrode rapidly [21].

5. Conclusion
The present study revealed that the potential electrogenic bacteria can be easily procured from waste water se-
diments and deployed in MFCs for an eco-friendly and economically viable method of electricity generation.
Bacterial isolates Z2 & Z7 can be further utilized to generate electricity from waste water. Bacterial isolate Z6
might prove to be potentially novel specie/genera and will be further investigated using conventional and mod-
ern microbial identification methods.

Acknowledgements
The present research has received FURAP Research Grant Funding from the Research Council of the Sultanate
of Oman (TRC). The authors would like to acknowledge support from TRC.

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