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Rainwater-driven microbial fuel cells for power generation in remote areas

Article in Royal Society Open Science · November 2021

DOI: 10.1098/rsos.210996?af=R

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Research
Cite this article: Amen MT, Yasin AS, Hegazy
Downloaded from https://royalsocietypublishing.org/ on 25 November 2021
MI, Jamal MAHM, Hong S-T, Barakat NAM. 2021
Rainwater-driven microbial fuel cells for power
generation in remote areas. R. Soc. Open Sci. 8:
210996.
https://doi.org/10.1098/rsos.210996
Received: 9 July 2021
Accepted: 29 October 2021
Subject Category:
Ecology, conservation and global change biology
Subject Areas:
energy/environmental engineering/
environmental chemistry
Keywords:
rainwater, microbial fuel cell, remote area sensor,
power generation
Author for correspondence:
Nasser A. M. Barakat
e-mail: nasbarakat@minia.edu.eg
Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.c.
5707013.
Rainwater-driven microbial
fuel cells for power generation
in remote areas
Mohamed Taha Amen1,4, Ahmed S. Yasin1,
Mohamed I. Hegazy4, Mohammad Abu Hena
Mostafa Jamal3, Seong-Tshool Hong3 and
Nasser A. M. Barakat2
1
Bio-Nanosystem Engineering Department, Chonbuk National University, Jeonju 561-756,
Republic of South Korea
2
Chemical Engineering Department, Faculty of Engineering, Minia University, Minia, Egypt
3
Department of Biomedical Sciences and Institute for Medical Science, Chonbuk National
University Medical School, Jeonju, Chonbuk, Korea
4
Microbiology Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt
MTA, 0000-0002-3623-1359
The possibility of using rainwater as a sustainable anolyte in an
air-cathode microbial fuel cell (MFC) is investigated in this
study. The results indicate that the proposed MFC can work
within a wide temperature range (from 0 to 30°C) and under
aerobic or anaerobic conditions. However, the rainwater
season has a distinct impact. Under anaerobic conditions, the
summer rainwater achieves a promised open circuit potential
(OCP) of 553 ± 2 mV without addition of nutrients at the
ambient temperature, while addition of nutrients leads to an
increase in the cell voltage to 763 ± 3 and 588 ± 2 mV at 30°C
and ambient temperature, respectively. The maximum OCP
for the winter rainwater (492 ± 1.5 mV) is obtained when
the reactor is exposed to the air (aerobic conditions) at
ambient temperature. Furthermore, the winter rainwater
MFC generates a maximum power output of 7 ± 0.1 mWm−2
at a corresponding current density value of 44 ± 0.7 mAm−2
at 30°C. While, at the ambient temperature, the maximum
output power is obtained with the summer rainwater (7.2 ±
0.1 mWm−2 at 26 ± 0.5 mAm−2). Moreover, investigation of
the bacterial diversity indicates that Lactobacillus spp. is
the dominant electroactive genus in the summer rainwater,
while in the winter rainwater, Staphylococcus spp. is the
main electroactive bacteria. The cyclic voltammetry analysis
confirms that the electrons are delivered directly from the
bacterial biofilm to the anode surface and without mediators.
Overall, this study opens a new avenue for using a novel
sustainable type of MFC derived from rainwater.
© 2021 The Authors. Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited.
 1. Introduction 2

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A microbial fuel cell (MFC) is a system in which the microorganisms can convert the chemical energy
embedded in some organic compounds to electricity through the oxidation of these compounds into
ATPs by sequential reactions [1–3]. The main difference between MFC and the other types of fuel cells is
the living microorganism acts as a catalyst and oxidizes the organic materials present at the anode
chamber [4–6]. Moreover, MFC is considered a clean, reliable and efficient process, and does not produce
any toxic by-products [7–9]. Although the energy produced by MFC is relatively low, it possesses a
unique feature in the fuel cells field; it can gain the chemical energy from several types of wastes
naturally present in different environments with potential and direct conversion into electrical energy.
Moreover, it can be operated at a pH close to neutrality and room temperature. Furthermore, MFC can
be considered the most convenient power-generating device for wireless sensors in remote areas [10,11].
Nowadays, wireless sensors are widely used in different fields such as environmental monitoring,
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homeland security, oceanographic study, medical applications and military tactical surveillance in remote

R. Soc. Open Sci. 8: 210996

locations for real-time data acquisition [12]. Sometimes, recharging or replacing the batteries for the sensor
is an impossible, time-consuming and/or impractical task. For resources-limited environments which
have no electricity grid or limited sunlight, MFC presents a promising power source for sensors
applications [8,13,14]. Moreover, the low power delivered by the MFCs is convenient for several types of
sensors, as 0.5 mW is sufficient for powering the autonomous sensor [13,15].
In this regard, development of a reliable MFC to power the autonomous sensors was achieved by
using sediments MFC (SMFC) [16,17]. Thomas et al. introduced a power management system (PMS)
based on a single sediment MFC [15], Zhang et al. compared two laboratory-scale SMFCs with a
difference in cathode arrangement (floating cathode versus bottom cathode) [18], Donovan et al.
developed an SMFC and a PMS to run a battery-less wireless sensor [12], and Shantaram et al.
designed an SMFC to operate electrochemical sensors and small telemetry systems to transmit the
data acquired by the sensors to remote receivers [19]. Indeed, the SMFC presents a perfect power
source for wireless sensors and other electronic devices, but it is still linked with the aquatic ecology
and cannot be exploited in many other places such as mountains and land areas.
Rainwater contains several kinds of microorganisms collected from the atmosphere. However, based
on our best knowledge, the rainwater was not previously investigated as self-containing exoelectrogens
anolyte to generate power through the MFC. The rainwater microbial fuel cell (RMFC) can be used at
any rainy place, so it could be a promising power generation device in the areas far from the aquatic
ecology. There is ample evidence for microbiological activity in the atmosphere [20]. Kaushik et al.
stated that the reservoir water and fresh rainwater samples exhibited a wide phylogenetic variety
including genomic sequences representing Alphaproteobacteria, Actinobacteria, Betaproteobacteria,
Gammaproteobacteria, Bacteriodetes and Lentisphaerae [21]. Recent studies have revealed the
presence of Escherichia coli, Pseudomonas aeruginosa, Klebsiella. pneumoniae and Aeromonas hydrophila
with different ratios in the examined rainwater samples [22]. These microorganisms have different
roles [23], moreover, there are viable microorganisms that are capable of using formate and acetate in
the atmosphere for their growth [20]. The bacteria are basic organisms in the atmosphere and play
important roles in the life cycle, and they are naturally washed from the air by rain. The presence of
microorganisms in the rainwater induced us to study the possibility of generating electricity through
RMFC because of their highly expected ability to metabolize organic/inorganic matters. In this study,
we investigate the exploitation of two different rainwater samples, based on sample collecting season,
as anolytes for electricity generation through a single chamber air-cathode MFC. The results are very
surprising as, although the rainwater was collected from a relatively high-quality atmosphere (Jeonju
city, South Korea) [24], a promising power output could be obtained.

2. Material and methods

2.1. Rainwater samples
The samples were collected in April and December for summer and winter rainwater samples,
respectively. 500 ml of each rainwater sample was collected in a sterilized glass bottle at an elevation
of 59 m.a.s.l. in Jeonju, Jeollabuk-do province, Republic of Korea. 200 ml from each sample
(immediately after collecting) was transferred to a 1 l sterilized bottle for microbiological (to
investigate the microbial community; §3.7) and bio-electrochemical (usability as anolyte in an MFC)
 analyses. These samples were transported in a cold box to the laboratory and processed within 8 h of 3
collection. The average corresponding electrical conductivities of the collecting samples were 61 and

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24 µS for the summer and winter rainwaters, respectively.

2.2. Anolyte preparation

The rainwater (without pretreatment) was used as anolyte without additives, and with Nutrient Broth
media (1 : 1 ratio v/v) which contains (for 1 l), 15 g of peptone (Samchun Pure Chemical Co., Ltd.),
3 g of yeast extract (Samchun Pure Chemical Co., Ltd.), 1 g of glucose (Junsei Chemical Co., Ltd.) and
6 g of sodium chloride (Samchun Pure Chemical Co., Ltd.), for comparison purposes.

2.3. MFC construction and operation

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A single chamber air-cathode MFC made of transparent polyacrylic was used as shown in
electronic supplementary material, figure S1. Four centimetres between the anode and the cathode was
maintained. An Ag/AgCl reference electrode is placed in the anode compartment to measure the
potential of the electrodes. Cation exchange membrane (CEM, CMI-7000, Membrane International Inc.,
NJ, USA) was used for ions exchange. The membrane was first treated by dipping in a H2SO4 solution
(0.5 M) for 18 h, and then kept in distilled water. The cathode was 2.4 cm × 2.4 cm Pt-loaded
(0.5 mgcm−2) carbon cloth (EC-20–5, Electro Chem, Inc., USA). The cathode material was in direct contact
with the natural air. A carbon felt (2.4 cm × 2.4 cm, 3.18 mm, Alfa Aesar) was used as an anode. The
power calculation was normalized for the anode projected surface area (6.25 cm2). The current collectors
were two 0.1 cm-thick high corrosion resistance stainless steel plates. The cell was assembled as described
in our previous work [25] as follows: the anode was attached to an anode current collector; this assembly
was placed at one side of the cell. Then, at the other side, the cathode was sandwiched between the CEM
and cathode current collector. The assembled MFC was sterilized by UV (CHC LAB Co., Ltd, Korea),
while the culture media and the glassware adopted in this study were autoclaved at 121°C for 20 min
before operation with a steam sterilizer (Autoclave, AC-60, Hanyang Scientific Equipment Co. Ltd, Korea).
To evaluate the performance, the RMFC was driven with pure rainwater as anolyte, and with a
mixture of rainwater and Nutrient broth media (1 : 1 ratio v/v). Furthermore, the aerobic and
anaerobic conditions were examined and the bacteria were cultivated under anaerobic conditions by
blocking the access of oxygen [26]. For the aerobic conditions, the MFC was in direct contact with the
ambient air through open access of 10 mm [27]. Typically, running the RMFC under anaerobic
conditions was performed by purging the anolyte using nitrogen gas bubbling for 5 min before using
and closing the anolyte feeding opening in the anolyte chamber in the assembled MFC. A photo
image can be found in the supporting information (electronic supplementary material, figure S1)
showing the closed inlet hole. On the other hand, aerobic conditions were applied by using the
anolyte without purging and opening the anolyte feeding opening; this strategy has been used
according to literature [26,27]. Moreover, the temperature effect was investigated, where the RMFC
was operated at the ambient temperature (0–5°C in January in Jeonju, South Korea) and at 30°C for
comparison purposes.

2.4. Electrochemical characterization

The MFCs’ electrochemical performances were evaluated using HA-151A potentiostat (HA-151A
POTENTIO STAT/GALVANOSTAT, Japan). The cyclic voltammetry (CV) analysis was carried out on
the assembled cell using a three-electrode set-up in which the anode was assigned as a working
electrode (WE), and the cathode and Ag/AgCl as a counter (CE) and reference (RE) electrodes,
respectively. The used potential window was from 1.0 to −0.4 V at a scan rate of 1 mVs−1. The open
circuit potential (OCP) values were recorded using GL220 midi-logger. After OCP stabilization, the
cell circuit was closed and the power curves were obtained by linear sweep voltammetry from the
maximum OCP to a zero-voltage at a scan rate of 1 mVs−1 with a two-electrode mode, where the
cathode was connected as WE and the anode as both CE and RE [28,29]. The power (P) was obtained
as P = IV. The morphology of the used anodes was investigated by scanning electron microscopy
(SEM Hitachi S-7400, Japan). The electrodes were dried at room temperature (20 ± 2°C) in sterilized
Petri dishes and then used for the analysis.
 2.5. Microbial community analyses 4

At the end of the experiment (after 22 days of operation), the RMFC was dismantled, and the anode was

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abstracted from the MFC in a sterilized laminar cabinet (CHC LAB Co., Ltd, Korea). The biofilm was
sampled from both anode surface and anolyte, and then cultured in Nutrient Agar media (Sigma-
Aldrich, Inc.). The biofilm was cultured to meet the DNA genomic isolation protocol requirements as
the extract DNA quantity should be enough for the identification step and this required enrichment
of the biofilm by culturing the biofilm [30]. The bacterial growth was subsequently isolated in a
separate culture using the same media according to the growth morphology characteristic. Finally, the
strains were grown overnight and processed to extract the total genomic DNA. The total genomic DNA
was isolated using DNA extraction kit protocol (QIAGene, Hilden, Germany) with a little modification
as described previously [30]. Briefly, 1 ml from all isolated cells were harvested in a 1.5 ml
microcentrifuge tube, after centrifuging for 5 min at 8000g. Then, the bacterial pellet re-suspended in
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180 µl buffer T1 and mixing well, followed by adding 25 µl proteinase K and vigorously homogenized

R. Soc. Open Sci. 8: 210996

by vortex and incubated at 56°C for 1–3 h with shaking during incubation. The samples after that were
vortexed and a volume of 200 µl of buffer B3 was added and mixed by vigorous vortexing. A 210 µl of
ethanol (100%) was added to the samples and vigorously mixed with vortex and then each sample was
applied to the Nucleospin Tissue Column and placed into a collection tube and centrifuged for 1 min at
11 000g. After discarding the flow-through from the previous step, a 500 µl of buffer BW was added
following by a centrifuge for 1 min at 11 000g. The Nucleospin Tissue Columns were placed in a new
collection tube, 600-µl buffer B5 was added and centrifuged for 1 min at 11 000g. The samples were
centrifuged again for 1 min at 11 000g. After that, the Nucleospin Tissue Columns were shifted to a
1.5 ml microcentrifuge tube and incubated for 1 min at room temperature after a 100 µl of prewarmed
buffer BE (70°C) was added. Finally, the previous tubes were centrifuged for 1 min at 11 000g and the
flow-through was kept at −4°C for the following analysis. The DNA purity ratio was determined using
260 and 280 nm absorbance ratios. The 16S rRNA gene was amplified using forward primer 27f
(50 -AGAGTTTGATCCTGGCTCAG-30 ) and reverse primer 1492r (50 -GGTTACCTTGTTACGACTT-30 ) by
polymerase chain reaction (PCR) as previously described [31]. 16S rRNA was used for the identification
of bacterial strains because it is a perfect universal target, and this gene was amplified using PCR to
increase the detection sensitivity of the band when running in an electrophoresis gel. Besides that, the
total bacterial count (TBC) was carried out using Nutrient agar media (Sigma-Aldrich, Inc.) [32] before
as well as after utilization in the MFC for comparison purposes.

3. Results and discussion

3.1. Season and temperature effects
Basically, it is expected that the kinds of microorganisms and their quantities in the rainwater will be vary
depending on the rainy season due to the change in temperature and other parameters. Therefore, the
samples were collected in the summer and winter seasons. As the proposed RMFC is supposed to
work within a wide temperature range through the year, so the investigation process was carried out
at the ambient conditions in winter (temperature range was 0–5°C) as well as at 30°C.
Using the summer and the winter rainwaters as anolytes revealed a noteworthy difference in MFCs
performance (figure 1 and electronic supplementary material, table S1). The summer rainwater MFC
(SRMFC) produced a maximum OCP (588 ± 0.8 mV) with 1.5 times more than that of the winter
rainwater MFC (WRMFC) at the ambient temperature after 15 days of operating time. Moreover, the
OCP needs a shorter time to start up in the case of the SRMFC than that for WRMFC (figure 1a). It is
noteworthy mentioning that both WRMFC and SRMFC reached almost a similar OCP after 20 days,
which may be explained as depletion of the nutrients in the two media. On the other hand, at a 30°C
working temperature, both of SRMFC and WRMFC, the maximum OCP (668 ± 0.9 and 763 ± 0.6 mV
for the WRMFC and SRMFC, respectively) was obtained after a relatively short time (three days)
compared to the ambient temperature. Furthermore, the OCP at 30°C was 1.2 and 1.7 times higher
than that at the ambient temperature for SRMFC and WRMFC, respectively (electronic supplementary
material, figure S2). Surprisingly, the OCP of WRMFC was superior performance compared to SRMFC
only during the period of 13–20 days of operation at 30°C (figure 1b).
Overall, the OCP results indicate that the proposed RMFCs can create a very acceptable potential
regardless of the collecting season and run at a relatively wide range of temperature degrees.
 (a) (b) 5
700 SRMFC SRMFC
800

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WRMFC WRMFC
600
700

500
600
voltage (mV)

voltage (mV)
400
500
300
400
200

100 300

200
0 5 10 15 20 25 0 5 10 15 20 25
time (days) time (days)
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R. Soc. Open Sci. 8: 210996

Figure 1. OCP for (a) SRMFC and WRMFC at ambient temperature; and (b) at 30°C.

(a) (b)
anaerobic SRMFC anaerobic WARMFC
aerobic SRMFC 600 aerobic WARMFC
600
voltage (mV)

voltage (mV)

400

400

200

200

0 5 10 15 20 25 0 5 10 15 20 25
time (days) time (days)

Figure 2. The OCP for (a) both anaerobic and aerobic reactors in the case of SRMFC, and (b) WRMFC.

Consequently, this finding suggests the possible application of the proposed RMFC in different weather
conditions. However, the remarkable differences between the OCPs of SRMFC and WRMFC conclude
that the summer rainwater is the favourable anolyte. The variation in the generated voltage could be
attributed to the differences in the microbial community. The bacterial community (species and
number) in the summer and winter rainwater has a large difference which in turn has a significant
impact on RMFC performance. The bacterial community variation is clear in the bacterial
identification besides the bacterial total count (see the Bacterial community analyses section).

3.2. Aerobic conditions effect

The proposed RMFC is suggested to work under pseudo-continuous mode in remote areas. In other
words, the future design of the RMFC will be based on an open-cell configuration having the
possibility of filling the cell with fresh rainwater to remove dead microorganisms and activating the
biofilm as well as providing nutrients. Moreover, the maintenance of anaerobic conditions in MFC is
not easy for real application [33]. Consequently, the performance of the introduced cells was
investigated under aerobic conditions. Overall, MFC is an anaerobic reactor because the oxygen is
acting as an inhibitor for the electroactive anaerobic microorganisms, but in the case of the facultative
electroactive bacteria (which are active with both aerobic and anaerobic conditions), the MFC could be
used under aerobic conditions [27]. As shown in figure 2a, in the case of SRMFC, the maximum OCP
was 471 ± 0.4 mV for the aerobic conditions compared to 588 ± 0.8 mV for the anaerobic state.
Interestingly, for the WRMFC, the aerobic reactor produces 492 ± 0.5 mV compared with 381 ± 0.8 mV
for anaerobic MFC (figure 2b). These results indicate that the main electroactive bacteria in the
SRMFC are highly active under anaerobic conditions, while in the case of the WRMFC, the main
electroactive bacteria are facultative alongside the anaerobic bacteria.
 (a) (b) 6
10 10 600
at 30˚C

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at 30˚C 800 9
with ambinet temperature 500

power density (mWm–2)

with ambinet temperature
power density (mWm–2)
8 700 8
600 7

potential (mV)
400

potential (mV)
6 500 6
5 300
400
4 4
300 3 200

2 200 2 100
100 1
0 0
0 0 20 40 60 80 100
10 20 30 40 50 60 70
current density (mAm–2) current density (mAm–2)
(c) (d)
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600 1.0

R. Soc. Open Sci. 8: 210996

0.18 at 30˚C at 30˚C
0.9 300
with ambinet temperature 500

power density (mWm–2)

with ambinet temperature
0.15 0.8
power density (mWm–2)

potential (mV)

potential (mV)
400 0.7
0.12
0.6 200
0.09 300 0.5
0.4
0.06 200
0.3 100
0.03 100 0.2
0.1
0 0
0 0.5 1.0 1.5 2.0 0 2 4 6 8 10
current density (mAm–2) current density (mAm–2)
(e) (f)
5
SRMFC SRMFC 700
4 600
WRMFC WRMFC
4 600
power density (mWm–2)

power density (mWm–2)

500

potential (mV)
500
potential (mV)

3
400 3
400
2 300 2 300
200 200
1 1
100 100
0 0
0 5 10 15 20 0
5 10 15 20
current density (mAm–2) current density (mAm–2)

Figure 3. Quasi-stationary polarization and power curves for (a) SRMFC after three days at ambient and 30°C, under anaerobic
conditions, (b) WRMFC after five days at 30°C and after 15 days for ambient temperature under anaerobic conditions, (c)
SRMFC after 22 days at ambient and 30°C under anaerobic conditions, (d ) WRMFC after 22 days at ambient and 30°C under
anaerobic conditions, (e) SRMF (after three days) and WRMFC (after seven days) at ambient temperature and under aerobic
conditions, and ( f ) SRMF and WRMFC after 22 days at ambient temperature and under aerobic.

3.3. Power curves

The successful role of the rainwater as anolyte in MFC could be properly determined by the MFC electricity
generation (figure 3). Comparatively, for SRMFC, a maximum output power of 7 ± 0.1 mWm−2 at 26 ±
0.5 mAm−2 and 7 ± 0.5 mWm−2 at 26 ± 0.5 mAm−2 was achieved under anaerobic conditions after three
days at ambient temperature and 30°C, respectively (figure 3a). On the other hand, the WRMFC
produced the maximum output power (2 ± 0.8 mWm−2 at 10 mAm−2) after 15 days when the reactor
was worked at ambient temperature, while the maximum output power (7 ± 0.6 mWm−2 at 44 mAm−2)
for 30°C was obtained after five days, under anaerobic condition (figure 3b). Subsequently, in the case of
SRMFC, the output power is slowly decreased to 1 ± 0.7 mWm−2 (at 9 ± 0.2 mAm−2) after 19 days when
the cell was run with ambient temperature, while the decrease was dramatic (1 ± 0.1 mWm−2 at
5 mAm−2) and fast (after eight days only) at 30°C working temperature (electronic supplementary
material, figure S3A). This finding can be attributed to the fast metabolization of the present nutrients at
 Table 1. Power generation (mWm−2) for the proposed RMFC working under anaerobic conditions and at ambient and 30°C. 7

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WRMFC SRMFC

time (day) ambient 30°C ambient 30°C

3 0.18 6 ± 0.41 7 ± 0.1 7 ± 0.5

5 0.15 7 ± 0.6 6 ± 0.2 2 ± 0.67
8 0.59 0.84 6 1 ± 0.1
15 2 ± 0.8 0.52 3 0.059
19 0.53 ± 0.03 0.48 ± 0.02 1 ± 0.7 0.04
22 0.72 ± 0.01 0.13 0.12 ± 0.01 0.035
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R. Soc. Open Sci. 8: 210996

30°C. While in the case of WRMFC, the power output was strongly affected after 19 days achieving 0.53 ±
0.03 mWm−2 at 4 ± 0.1 mAm−2 and 0.48 ± 0.02 mWm−2 at 2 ± 0.1 mAm−2 for the ambient and 30°C
temperature, respectively, (electronic supplementary material, figure S3B).
Finally, after 22 days, the output power was limited to 0.12 ± 0.01 mWm−2 at 0.85 ± 0.02 mAm−2
and 0.035 mWm−2 at 0.22 ± 0.02 mAm−2 for SRMFC at ambient and 30°C temperatures, respectively,
(figure 3c). Whereas in the case of the WRMFC, the output power was 0.72 ± 0.01 mWm−2 at 6 ±
0.1 mAm−2 and 0.13 mWm−2 at 1 mAm−2 at ambient and 30°C temperatures, respectively (figure 3d).
Interestingly, under aerobic conditions and at ambient temperature (figure 3e,f ), the maximum power
output for the WRMFC was achieved after seven days (3 ± 0.49 mWm−2 at 7 ± 0.2 mAm−2) which in turn
was two times more than the maximum output power (1 ± 0.74 mWm−2 at 7 ± 0.2 mAm−2) for SRMFC
after three days (figure 3e). Furthermore, after 22 days, the SRMFC output power was limited to
0.15 mWm−2 at 1 mAm−2, while the WRMFC output power is sustained close to the maximum power
(3 ± 0.3 mWm−2 at 10 ± 0.3 mAm−2; figure 3f ).
The output power values emphasize that the RMFC can be used with either summer or winter
rainwater as anolyte and under both aerobic and anaerobic conditions. Moreover, these results show
that the season of the anolyte collection, as well as the operation conditions (temperature degree and
aerobicity), have a distinct influence on the MFC output power. The summer rainwater is the
favourable anolyte with the ambient temperature where it achieved output power higher than that for
the winter rainwater with 2.5 times (around 7 and 3 mWm−2, respectively). On the contrary, the
winter rainwater is the proper anolyte in the case of the aerobic condition, where the WRMFC power
output was 22-fold higher than the power output for the SRMFC (around 3 and 0.15 mWm−2,
respectively). Tables 1 and 2 summarize the obtained powers from the proposed RMFCs.
Although, it seems that the generated power from the proposed RMFCs is small, however using the
rainwater without inoculation strongly recommends exploiting these cells as power sources in the remote
areas as duplicating the power can be achieved by assembling the individual cells in a stack. Moreover,
the successful running of the proposed microbial fuel cells under aerobic conditions changes the
proposed cells to be a sustainable source of power. Simply, if the constructed cells were left open to
the atmosphere, the cells will be loaded by fresh rainwater which changes the system to be a
continuous power source.

3.4. Reliability evaluation

The addition of nutrients to enrich the rainwater flora is possible in laboratory experiments, but it is a
difficult task in real applications. Therefore, the summer rainwater (alone without additives) was
invoked to evaluate the feasibility of using a pristine rainwater as anolyte in the proposed MFC at an
ambient temperature in the winter (0–5°C).
Interestingly, the rainwater with and without nutrients showed almost the same progression with
close maximum voltage output (figure 4a). The summer rainwater alone (SRMFC-A, for short)
produced a maximum voltage output of 553 ± 0.6 mV, with only a 50 mV decrease compared to the
maximum output of the summer rainwater with nutrients (588 mV). Furthermore, the OCP
progression rate of summer rainwater with nutrients (SRMFC-WN, for short) needs a short time to
start up and achieve the maximum output OCP (six and 16 days, respectively), while SRMFC-A
required more than 14 days to start up, where the maximum OCP was achieved at 18 days.
 (a) (b) 8
8
SRMFC with nutrients SRMFC with nutrients
700

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SRMFC without nutrients 7 6 days SRMFC without nutrients
9 days decline

power density (mWm−2)

600 6
voltage (mV)

5
500
4
400
16 days 3

300 2 18 days
1
200

0 5 10 15 20 25 0 5 10 15 20 25
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time (days) time (days)

R. Soc. Open Sci. 8: 210996

Figure 4. The SRMFC with and without nutrients, (a) voltage output, and (b) power density, throughout the operation period.

Table 2. Influence of aerobic condition on the generated (mWm−2) for the proposed RMFC at ambient temperature.

time (day) WRMFC SRMFC

3 2 ± 0.2 1 ± 0.74
7 3 ± 0.49 0.34
22 3 ± 0.3 0.15

In the case of the SRMFC-A, the maximum power output (2.44 ± 0.05 mWm−2 at 10 ± 0.5 mAm−2)
was observed after 18 days, while the SRMFC-WN achieved the maximum power output
(7 ± 0.1 mWm−2 at 26 ± 0.5 mAm−2) after three days (electronic supplementary material, figure S4B).
The short startup of RMFC-WN could be ascribed to the effect of nutrients regarding the bacterial
growth curve [34], where the enrichment of rainwater accelerated the microbial flora growth rate to
finish the lag phase and start the log and then stationary phases in a short time. This acceleration
influence appeared to be a passive effect for sustaining SRMFC-WN, whereas the stationary phase
starts rapidly, it has been also terminated rapidly and the OCP and power output declined steadily
after achieving their maximum outputs directly (at 16 and nine days of operation, respectively).
Therefore, the SRMFC-WN sustained the voltage output at the same rate for nine days and just six
days for power generation through the operation period (figure 4b and electronic supplementary
material, figure S4A). Overall, these results revealed that the SRMFC alone without nutrients could
produce a reasonable voltage output with a wide range of temperature degrees, and the possibility of
the proposed RMFC in a real application accordingly.

3.5. Cyclic voltammetry

The CV analysis can be exploited as a powerful methodology to check the dependency of the electron
transfer on mediators [35]. Briefly, the mediators (also known as shuttles) are considered shuttle
carriers for the electrons from the microorganism surface to the anode. The electron transfer takes
place in the form of redox reactions. In other words, the mediators are reduced at the microorganism
surface and oxidized at the anode. Therefore, the presence of redox peaks in the CV cycle can be
assigned to the presence of mediators [36,37]. Cyclic voltammograms of the RMFC were carried out at
1 mVm−1 scan rate. As shown in figure 5, smooth and peaks-free cycles were obtained for both
SRMFC and WRMFC. Accordingly, it can be claimed that the electrons are delivered from the
bacterial biofilm to the anode surface by direct contact and without mediators [2].

3.6. Scanning electron microscopy (SEM) analysis

SEM analysis (figure 6) was carried out at the end of the experiments to check the bacterial biofilm
morphology on the anode surface. In general, a thick and good-structured biofilm could be observed
 600.0 m SRMFC 9
WRMFC
400.0 m

royalsocietypublishing.org/journal/rsos
200.0 m

current (A)
–200.0 m

–400.0 m

–600.0 m

–800.0 m

–1.0 m
Downloaded from https://royalsocietypublishing.org/ on 25 November 2021

–0.4 –0.2 0 0.2 0.4 0.6 0.8 1.0 1.2

potential (V)

R. Soc. Open Sci. 8: 210996

Figure 5. CV curves for the SRMFC and WRMFC at 1 mVs−1 scan rate after OCP stabilization.

(a) (b)

Figure 6. SEM images for bacterial biofilm for both; (a) SRMFC and (b) and WRMFC.

for both SRMFC and WRMFC, and there were no fundamental differences between their biofilm’s
appearance. These images revealed that the rainwater flora contains electroactive bacteria that
successfully attached closely to the carbon felt’s thread surface and developed an electroactive biofilm
which in turn transmits electrons to the anode.
Biofilm uniformity at the anode surface indicates a non-fermentable fuel feeding [38], while
fermentable fuel feeding is associated with microbial clumps appearance in the anodic biofilm [39]. The
uniformity of observed biofilm of both SRMFC and WRMFC represent a non-fermentative electroactive
bacteria, where rainwater contains simple compounds (formate, acetate, etc.) which fermentation needs
before microbial community consumption [40].

3.7. Bacterial community analyses

3.7.1. Bacterial population
Before using in the RMFCs, the as-collected rainwater samples have been examined using
microbiological analyses to investigate the microbial community in the used rainwater. The TBC is
showing different bacterial loads as presented in table 3. The variation in the TBC is related to the
sampling season (summer and winter). TBC clarified that the bacterial load in the winter rainwater
sample is less than that in the summer one which in turn could be an acceptable reason alongside the
bacterial species for the variation in the power output between the two samples.

3.7.2. Bacterial diversity of RMFC

Synergetic interactions between microorganisms present an important role in the formation of
electroactive biofilms, which in turn, is the main key in the longevity and success of bio-
electrochemical systems [41]. The sequences with the similarity ratio to strains in both SRMF and
 Table 3. The TBC for the summer and winter rainwater samples before and after using with MFC. 10

royalsocietypublishing.org/journal/rsos
before using after using

summer rainwater winter rainwater SRMFC WRMFC

5.14 × 103 4.19 × 103 8.12 × 103 7.13 × 103

Table 4. Identification of 11 DGGE bands based on 16S rRNA genes, from the anode surface biofilm and anolyte.

band closest sequence similarity (%) accession no. cited isolation source

1 Lactobacillus coryniformis strain 17-9 99 NR_029018.1 summer rainwater

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R. Soc. Open Sci. 8: 210996

2 Lactobacillus coryniformis strain DPP-LP3 93 NR_029018.1 summer rainwater
3 Lactobacillus coryniformis strain KLDS 1.0723 99 NR_029018.1 summer rainwater
4 Bacillus cereus strain P2 99 NR_074540.1 summer rainwater
5 Bacillus methylotrophicus strain CBMB205 99 NR_116240.1 summer rainwater
6 Lactobacillus coryniformis strain 17-9 99 NR_029018.1 summer rainwater
7 Staphylococcus capitis strain Y85XJ04 95 NR_113348.1 summer rainwater
8 Staphylococcus capitis strain ATCC 27840 99 NR_117006.1 summer rainwater
9 Staphylococcus capitis-Y41 99 JX_094948.1 winter rainwater
10 Staphylococcus capitis strain BQEN3-03 98 FJ_380955.1 winter rainwater
11 Enterobacteriaceae bacterium strain 4 97 KY_681857.1 winter rainwater
12 Staphylococcus capitis strain 309-16 98 MG_557816.1 winter rainwater
13 Staphylococcus capitis strain BBN3T-04d 98 FJ_357614.1 winter rainwater
14 Pseudomonas sp. CSJ-3 98 KF_861966 winter rainwater

WRMFC is presented in table 4. It is clear that Lactobacillus sp. (electronic supplementary material,
table S2) is the main electroactive bacteria in SRMFC, which was previously stated as electroactive
bacteria in the MFC application [42]. Besides, Bacillus sp. and Staphylococcus sp. are present in the
SRMFC bacterial community, and their role as the electroactive effect was also previously reported
[41,43]. However, Staphylococcus sp. (electronic supplementary material, table S3) is the dominant
genus and main electroactive in the case of the WRMFC. Furthermore, Pseudomonas spp., which
is well known as an electroactive genus in MFC45, was detected in the WRMFC. Moreover, the
Enterobacteriaceae bacterium strain was also found in the investigated sample, however, its role as
electroactive bacteria still needs more study.
Overall, the proposed MFC is applicable in many countries due to the high average raining days
around the year based on the world weather and climate website [44]. Electronic supplementary
material, figure S5 shows the average daily water in some randomly selected countries, however, on
the website, the average of rainy days could be found for many countries. Practically, the proposed
RWMFC can work in the remote area by first filling the cell with the rainwater and leaving it at open
circuit mode until the formation of the biofilm at the anode surface and stability of OCV. Later, any
additional rainwater that fills the cell will be used as a source of nutrients for the microorganisms to
generate electricity.

4. Conclusion
Rainwater can be effectively exploited as an anolyte in an air-cathode single-chamber MFC. Based on the
results, it is preferable to use collected water within the summer season for the anaerobic conditions
MFC. On the other hand, winter rainwater is recommended for aerobic reactors. In the case of the
summer rainwater reactor, the dominant electroactive bacteria were Lactobacillus spp. However, the
main electroactive genus in the winter rainwater reactor was Staphylococcus spp. The investigated
 rainwater-driven microbial fuel cells might be used not only for powering wireless sensors but also as a 11
biosensing tool to monitor the atmosphere microbial community and the ratio of different elements and

royalsocietypublishing.org/journal/rsos
their toxicity [36]. Based on our best knowledge, the rainwater was not previously investigated as anolyte
for the MFC; consequently, this area needs more investigation to adopt this valuable source of energy and
overcome the obstacles.
Data accessibility. Data are available from the Dryad Digital Repository: http://doi.org/10.5061/dryad.pvmcvdnm7 [45].
The data are provided in the electronic supplementary material [46].
Authors’ contributions. M.T.A. carried out the laboratory work, participated in data analysis, carried out sequence
alignments, participated in the design of the study and drafted the manuscript; A.S.Y. participated in the statistical
analyses; M.I.H. and M.A.H.M.J. participated in the biological study; S.-T.H. participated in the biological study
and discussed their results; N.A.M.B. conceived of the study, designed the study, coordinated the study and helped
draft the manuscript.
Competing interests. We declare we have no competing interests.
Downloaded from https://royalsocietypublishing.org/ on 25 November 2021

Funding. We received no funding for this study.

R. Soc. Open Sci. 8: 210996

Acknowledgements. The authors wish to thank the Royal Society Open Science Editorial Office, associate editors, and the
anonymous reviewers for the quality of their feedback and very insightful comments that have contributed to the final
version of the paper.

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