Environmental Science and Pollution Research

https://doi.org/10.1007/s11356-021-15104-w

RESEARCH ARTICLE

Application of rotten rice as a substrate for bacterial species

to generate energy and the removal of toxic metals from wastewater
through microbial fuel cells
Najwa Najihah Mohamad Daud 1 & Akil Ahmad 2 & Asim Ali Yaqoob 1 & Mohamad Nasir Mohamad Ibrahim 1

Received: 8 March 2021 / Accepted: 21 June 2021

# The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
Microbial fuel cells (MFCs) are the efficient and sustainable approach for the removal of toxic metals and generate energy
concurrently. This article highlighted the effective use of rotten rice as an organic source for bacterial species to generate
electricity and decrease the metal concentrations from wastewater. The obtained results were corresponding to the unique
MFCs operation where the 510 mV voltage was produced within 14-day operation with 1000 Ω external resistance. The
maximum power density and current density were found to be 2.9 mW/m2 and 168.42 mA/m2 with 363.6 Ω internal resistance.
Similarly, the maximum metal removal efficiency was found to be 82.2% (Cd), 95.71% (Pb), 96.13% (Cr), 89.50% (Ni), 89.82
(Co), 99.50% (Ag), and 99.88% (Cu). In the biological test, it was found that Lysinibacillus strains, Chryseobacterium strains,
Escherichia strains, Bacillus strains are responsible for energy generation and metal removal. Furthermore, a multiparameter
optimization revealed that MFCs are the best approach for a natural environment with no special requirements. Lastly, the
working mechanism of MFCs and future recommendations are enclosed.

Keywords Microbial fuel cell . Organic substrate . Rotten rice . Metal removal . Energy generation

Introduction
Microbial fuel cells (MFCs) have been recognized as a poten-
tial bioelectrochemical technique. It can convert the chemical
energy into electrical energy by catalytic activities of micro-
organisms (Logan et al. 2006). In this technique, bacterial
metabolism can provide energy in the form of an electric cur-
rent. In the field of MFCs, research has led to increasing con-
cern and a dramatic increase in the number of publications.
The MFCs system has been considered as a sustainable, safe,
and efficient production of bioelectricity (Yaqoob et al.
Responsible Editor: Weiming Zhang
* Asim Ali Yaqoob
asimchem4@gmail.com
* Mohamad Nasir Mohamad Ibrahim
mnm@usm.my
1
Materials Technology Research Group (MaTRec), School of
Chemical Sciences, Universiti Sains Malaysia, 11800 Minden,
Penang, Malaysia
2
Centre of Lipids Engineering and Applied Research, Universiti
Teknologi Malaysia, UTM, 81310 Skudai, Johor, Malaysia
2020a; Yaqoob et al. 2020b). The electron and proton are
produced as the result of the organic substrate oxidation pro-
cess. Several organic wastes such as domestic wastewater,
municipal wastewater, and excess sludge were used in earlier
studies (Asefi et al. 2019; Jia et al. 2013; Yaqoob et al. 2021a).
Generally, substrates such as carbohydrates are used in an
anaerobic anode chamber. According to Li et al. (2016) sub-
strates are considered as one of the most significant biological
factors in MFCs that influence electricity generation. The sub-
strates affect the integral composition of the bacterial popula-
tion at the anode biofilm as well as the power density (PD) and
pollutant removal efficiency in the performances of MFCs.
Recently, sustainable energy generation and pollutant removal
via MFCs by using food wastes have gained much attention
due to the large production of food waste. Several studies
showed that food waste is feasible as the organic substrate to
produce electricity in MFCs (Asim et al. 2021, Goud et al.
2011, Moharir and Tembhurkar 2018, Rikame et al. 2012).
For example, Li et al. (2016) used canteen based food waste as
organic substrate in MFCs and achieved a maximum power
density of 5.6 W/m3 and average output voltage of 0.51 V. It
was concluded that the recovery of electricity was achieved by
using food waste as substrates in MFCs, however, it is quite
 Environ Sci Pollut Res

challenging for the enormously complicated process structure nickel nitrate hexahydrate (R & M Chemical), cobalt nitrate
of the combination of hydrolysis, methonogenesis, hexahydrate (R & M Chemical), silver nitrate (R & M
acidogenesis, and acetogenesis. Thus, the pre-treatment pro- Chemical), copper nitrate (R & M Chemical), etc. Distilled
cesses such as sonication and microwave pre-treatment were water was used throughout the study.
often required (Xin et al. 2018; Yaqoob et al. 2020c; Yusoff
et al. 2013). In Malaysia, rice is the most consumed food
(twice or thrice) in a day. In 2018, Solid Waste Management Inoculation source
and Public Cleansing Corporation (SWCorp) has reported that
wasted food daily mostly rotten rice generated from the house- The pond wastewater from Tasik Fajar Harapan, Universiti
hold followed by night markets, food courts, hotels, and Sains Malaysia was collected and treated with 50 ppm of
school canteens are the highest 44.5% and this amount rises different toxic metal ions. The metal-containing wastewater
by 15 to 20% during festive seasons (Azhari 2019). The was labeled as synthetic wastewater in the present study and
amount of rice particularly in Malaysia is adequate to feed finally used as a source of inoculation in MFCs. Different
12 million people three times a day and the average physicochemical parameters for pond wastewater and synthet-
Malaysian throws away is 1.64 kg per day relative to the world ic wastewater are shown in Table 1. About 800g of rotten rice
average of 1.2 kg per day (Azhari 2019; Goud and Mohan waste material was used as an organic substrate for bacterial
2011). This concern has created serious environmental pollu- activities. The pH, temperature, and conductivity were deter-
tion problems and health threats when much of the rotten rice mined using pH meter (EUTECH instrument-700; New York,
today is disposed of by conventional methods such as landfill, NY, USA), thermometer (GH, ZEAL LTD; London, UK),
composting, and incineration, which may contribute to pollu- and electrical meter (ECM) (Alpha-800 conductivity meter,
tion of groundwater, the attraction of vermin, production of Vernon Hills, IL, USA).
poisonous gasses, and odor problems. Moreover, convention-
al approaches are inefficient and impracticable because they MFCs design and set-up
cannot be entirely or efficiently used as desirable nutrients and
energy sources from food waste, such as rotten rice. In this study, a single chamber of MFCs was used. The MFCs
Therefore, using the rotten rice as a substrate in MFCs for a were 16.2 cm in height and had a diameter of 13.4 cm. The
bioenergy recovery viewpoint could be an ideal solution for a total working volume of the MFC was 1000 mL but 800 mL
solid waste management. According to our best knowledge, prepared synthetic wastewater was inoculated with 800 g of
the use of food waste as an organic substrate for the growth of rotten rice in a single chamber of MFCs. Next, the commercial
the bacterial community is new in MFCs. In this present study, graphite rods of 14.9 cm x 1cm (h x r) anode and 9.5 cm x
rotten rice collected from a cafe in Universiti Sains Malaysia 1 cm (h x r) cathode were vertically embedded into the MFCs.
and has been utilized as anodic substrate in a single chamber The gap between anode and cathode was 17.5 cm. The elec-
MFCs for direct energy generation. Additionally, we also car- trodes were connected by using platinum wire in the presence
ried out the removal of Cd2+, Pb2+, Cr3+, Ni2+, Co2+, Ag+, and of 1000Ω external resistance. MFCs experiment of rotten rice
Cu2+ from synthetic wastewater. The aim to monitor the re- was operated for 30 days at the room temperature range from
moval performance is to highlight the effect of organic sub-
strate (rotten rice) toward the removal efficiency. The obtained Table 1 Parameters of the pond and synthetic wastewaters utilized in
findings have been well confirmed by various electrochemical the present study
tests (replication processes were performed) and biological Parameters Pond wastewater Synthetic wastewater
characterizations.
Color Light yellow Dim yellow
Electrical Conductivity 24μs/cm 118 μs/cm
Experimental details Temperature 25 ± 2 °C 25 ± 2 °C
pH 6.91 6.26
Chemicals and materials Odor Bad smell Bad smell
Cd2+ 0 ppm 50 ppm
Rotten rice waste material was collected from a café in Pb2+ 0 ppm 50 ppm
Universiti Sains Malaysia. The pond wastewater was collected Cr3+ 0 ppm 50 ppm
from Tasik Fajar Harapan, Universiti Sains Malaysia. Various Ni2+ 0 ppm 50 ppm
materials have been procured from different sources namely Co2+ 0 ppm 50 ppm
commercial graphite rods (FUDA 2B Lead, NY, USA), cad- Ag+ 0 ppm 50 ppm
mium nitrate tetrahydrate (R & M Chemical), lead nitrate (R Cu2+ 0 ppm 50 ppm
& M Chemical), chromium (III) nitrate (R & M Chemical),
Environ Sci Pollut Res
25 °C to 30 °C. The model of MFCs and used reactor in the
present study are shown in Fig. 1.
Electrochemical tests
The potential voltage between anode and cathode was moni-
tored one time in 24 h by using a digital multimeter (UNI-T
UT33A, China). Ohm’s law was used to interpret voltage into
current (Ampere). To calculate the power density (PD), cur-
rent density (CD), and internal resistance (r), the Eqs. (1-4)
were used where voltage output was denoted as V, the current
was denoted as I, cross-sectional area was denoted as A, in-
ternal resistance was denoted as r and electromotive force was
denoted as E.
Voltage Output ðVÞ ¼ IR
V2
Power Density ðPDÞ ¼
RA
I (EIS; Gamry Reference 600; Warminster, PA, USA) was con-
Current Density ðCDÞ ¼
A ducted at a different time interval to analyze the resistance
 
E−V
Internal Resistance ðrÞ ¼ R ð4Þ
V
The electromotive force was measured via an open circuit
voltage (OCV). The slope of the polarization curve involved a
variable resistance box from 7000 Ω to 100 Ω was used to
determine the internal resistance of MFCs.
Cyclic Voltammetry (CV) and Electrochemical
Impedance Spectroscopy (EIS)
The redox reactions occurring on the electrode surface were
characterized by using cyclic voltammetry (CV, Model BAS
Epsilon Version 1.4; West Lafayette, IN, USA). The electrode
Fig. 1. a A schematic diagram of MFCs b The actual MFCs set-up for rotten rice waste used in the present study
surface was measured at a 30 mV/s scanning rate with a po-
tential range between +0.8V to −0.8V at 10th, 20th, and 30th of
time intervals. The platinum wire and glassy carbon were used
as an electrode counter and operating electrode respectively,
with Ag/AgCl as the reference electrode. The electrode poten-
tial was determined against the reference electrode. The spe-
cific capacitance, Cp (F/g) is defined as the integrating both
anode and cathode through the entire set of data per unit area
of the cathode and anode. Cp was calculated using the data
derived from the CV calculation according to the following
Eq. (5) where the area of CV curve was denoted as A, loaded
sample quantity in CV instrument was denoted as m, the scan
rate of CV in mV/s was denoted as k, and potential range of
CV was denoted as (V2 − V1).
ð1Þ A
Cp ¼ ð5Þ
2mk ðV2−V1Þ
ð2Þ
Further, the Electrochemical Impedance Spectroscopy
ð3Þ
effect of anode toward voltage. On day 10th, 20th, and 29th
EIS analysis was performed in the operative mode of MFCs
from a frequency range of 100 kHz to 100 mHz. The ampli-
tude of alternating current (AC) was estimated at 1 mV to
avoid the attachment of biofilm and to minimize the disruption
of the steady-state system.
Removal of toxic metal ions
For the removal performance, toxic metal ions contents were
analyzed using an atomic absorption spectrometer (AAS;
PerkinElmer AAnalyst 400; Waltham, MA, USA). Every 10
days, about 1 mL of sample was collected from the MFCs and
analyzed the metal contents. Equation (6) was used to

calculate the percentage of removal efficiency (RE %) where
the initial concentration of metal ions was denoted as Minitial
and the final concentration of metal ions after MFCs operation
was denoted as Mfinal.
Minitial −Mfinal
Removal efficiency% ¼  100 ð6Þ
Minitial
Biological characterizations
Scanning electron microscopy (SEM-Zeiss, DSM-960,
Germany) was performed to study the growth, surface
morphology, and stability of the biofilm. The SEM images
verify the bacterial growth on the surface of the anode
where anode was subjected to the removal of metal con-
centrations from synthetic wastewater. The energy disper-
sive X-ray (EDX) of the biofilm-anode electrode was taken
at the end of the operation to study the effect of the toxic
metals on the biofilm (Biofilm is a cluster of bacterial spe-
cies). Further, in bacterial isolation and identification,
about 1.00 mm of biofilm formed was scratched from the
anode surface. At the end of the MFCs process, the biofilm
was stored in distilled water. The colonies on nutrient agar
plates were transferred by using a serial dilution method.
After some time, nutrient agar plates showed a variety of
colonies. They are being purified and analyzed very care-
fully to identify the bacterial species. Then, pure cultured
nutrient agar plates were kept in a fridge until the measure-
ment was carried out. The polymerase chain reaction
(PCR) method was used to produce bacterial 16S rRNA
genes. Amplification of the genes was achieved by using
a forward primer (27F) and reverse primer (1492R). The
PCR amplified product was cloned with the use of a clon-
ing kit (TOPOTA, Invitrogen; Carlsbad, CA, USA). After
DNA sequencing analysis, 16S rRNA bacterial strains
were deposited in GenBank.
Multiple parameter optimizations
In this study, a series of experiments have been carried out to
optimize the different parameters such as pH, temperature, en-
ergy efficiency, and metal removal. The optimized parameters
were carried out to evaluate the MFCs ' performance and were
supposed to have greater energy efficiency as well as metal
removal which is an indicator of change in the metabolic phase
of the bacterial community. Each parameter was analyzed by
using the commercial graphite as anode and cathode with 1000
Ω external resistance. Rotten rice as an organic substrate was
used for all the optimization standards in this experiment. The
inoculated source was 800 mL of synthetic wastewater which
contained Ag, Cu, Cd, Cr, Co, Ni, and Pb metal ions. The
temperature ranges from 20, 30, to 35 oC were analyzed. The
Environ Sci Pollut Res
voltage reading was measured after 48 h at each temperature and
the removal efficiency was calculated on day 5. The pH of the
inoculation source was set to 7 during the temperature optimi-
zation. Further, pH range 4, 5, 6, 7, 8, 9, and 10 were carried out.
The voltage reading was taken after 24 h on each pH range and
the removal efficiency was calculated on day 5. The pH of the
inoculation source was adjusted by using H2SO4 and NaOH.
The room temperature was used to optimize the pH range.
Results and discussion
Voltage trend, polarization behavior, and internal
resistance measurement
The experiment was successfully carried out for continued 30-
day operation by using the rotten rice as an inoculation source in
MFCs. From Fig. 2b, the operation was continued for three
cycles to achieve the maximum voltage generation. The maxi-
mum achieved voltage was 510 mV (0.510 mA) on the 14th day.
While on completion of the first cycle the voltage started declin-
ing until 234 mV (0.234 mA). It might be due to the completion
of the life cycle of the bacterial species. After a certain period, it
started again increasing the trend of voltage and reached up to
380 mV but still, it did not deliver more than the value recorded
in the first cycle. Similarly, in the 3rd cycle, the observation was
lower than the second and third cycle. Finally, the voltage con-
stantly started decreasing after a 30-day operation which is an
indication of the operation completion. The observed voltage
under the open circuit was 750 mV. The present results are quite
interesting in the field of MFCs as compared to previous litera-
ture. For example, Yaqoob et al. (2020b) follow a similar trend
and achieved a maximum of 200 mV in 36 days by utilizing the
plant-extract sap as organic substrate. It means that the higher
voltage trend found in the present study due to the stable inoc-
ulation source (rotten rice). Further, the voltage generation trend
also indicates the removal efficiency rate, on the completion of
the first cycle indicating that the Cd2+ was converted into Cd(s).
To continue operational cycles, it was showing the turning point
of conversion of the soluble states to insoluble states. Further, to
study the PD, CD, and voltage relationship by varying the ex-
ternal resistance, polarization studies were carried out. The 7000
Ω to 100 Ω resistors were connected one by one during the
continuous MFCs operation. The observation showed that high
external resistance displayed low electron transportation due to
the electronic resistance and high destabilization. While the low
external resistance showed less stability in the electronic move-
ment due to rapid transference of electrons. The external resis-
tance and internal resistance of the cell should be comparable for
feasible electronic movement without ohmic resistance. To find
the appropriate fixed external resistance, before the operation, a
series of external resistance was attached. At high external resis-
tance, the voltage decreased from the open-circuit voltage
Environ Sci Pollut Res
Fig. 2. a Voltage trend b polarization curve c CV curves d EIS curve at different time interval
(OCV) and later gradually recovered. However, during the ex-
ternal resistance variation, the cell design point was found at 500
Ω. The maximum achieved PD and CD were 2.9 mW/m2 and
168.42 mA/m2, respectively. The observed internal resistance
was 363.6 Ω. It is also indicating that the higher external resis-
tance of more than 500 Ω can decrease electron transportation.
Therefore, the external resistance of 500 Ω can deliver the max-
imum energy output as compared to the higher resistance.
During the observation, it was observed that 0.5 mW/m2 PD
at 7000 Ω while 100 Ω delivered a maximum of 1.2 mW/m2
PD. It means the electronic resistance is also a factor that needs
attention to control the electron resistivity without destabiliza-
tion. The internal resistance can be decreased by reducing the
distance of the anode and cathode anode. That is why several
studies preferred to use single-chamber MFCs instead of double
chamber MFCs (Chae et al. 2010; Do et al. 2018; Du et al.
2007).
Cyclic voltammetry, specific capacitance, and
Electrochemical Impedance Spectroscopy studies
Figure 2c showed the measured CV curves at different time
intervals to study the electronic mobility during the MFCs oper-
ation. The CV curves showed the current in the forward scan (FS)
and reverse scan (RS) at different days. On day 10 the FS was
1.5×10-5 mA, day 20 (2.9 ×10-5 mA) and day 30 (4.5×10-5 mA)
while the RS was on day 10 (−0.5×10-5 mA), day 20 (1.2 ×10-5
mA) and day 30 (−1.80×10-5 mA). The maximum FS and RS
were observed on day 30. It means that the rate of oxidation of
organic substrate and reduction was high which increased gradu-
ally and reached a maximum on the 30th day. The maximum
oxidation of the present study showed 0.8 mA and the reduction
rate was −0.8 mA. Overall, the CV showed the maximum oxida-
tion and reduction rate throughout the operation. Due to the rotten
rice as inoculation, the reaction was quite fast as compared to
previous studies (Yaqoob et al. 2020b; Yaqoob et al. 2021a).
Additionally, the CV curves are also studied to calculate the
Cp values. The Cp values showed the formation rate and stability
of the biofilm throughout the operation. According to the CV
curves, we found that the biofilm was gradually produced and
showed good stability with rotten rice inoculation source.
Usually, the low value of Cp indicates that the biofilm growth
is in process or a less stable but gradually increased value
indicating high stability of biofilm on the anode. Table 2
showed the Cp value of the present study which is showing the
high performance of biofilm. Hong et al. (2011) also used a
similar concept to describe the formation rate and stability of
the biofilm via CV curves.

Table 2 Biofilm assessments through CV curves via calculating the Cp
at different intervals of operation
Measurement time interval Capacitance (F/g)
10th 3×10-5
20th 9×10-5
30th 14×10-4
Figure 2d showed the EIS- Nyquist plots to study the
charge transfer resistance by using the equivalent circuit mod-
el. The EIS measurement was carried out at a different time
interval to analyze the operation. Initially, the biofilm was
gradually produced and on the 10th day, the fuel cell was
operated with EIS-potentiostat. Generally, EIS- Nyquist plots
correspond to the lower electronic mobility if the straight line
comes with high Z‘img (Ohm), while semicircle or semibent
with low Z‘img (Ohm) which correspond to the high mobility
of electrons (Hung et al. 2019). The results on the 10th day
showed the straight line which indicated that the charge resis-
tance was higher as compared to mobility. Later, on the 20th
day, the improved high electronic mobility was observed with
bent curves which indicated that the biofilm is fully developed
and stable. The electrons are effectively moving toward the
cathode. Similarly, on the 29th day also showed excellent
electronic movement with 363.6 Ω internal resistance.
Usually, at lower internal resistance the charge resistivity is
low which allows the electrons to move more easily.
Metal removal trend and bacterial identification
studies
Nowadays, metal ions removal through MFCs is an emerging
area. Recently, the idea to use the waste-derived inoculation
source to produce electricity and decrease the metal ion
concentration is the most emerging area of study. In the
present study, the metal removal using the MFCs via rotten
rice as an inoculation source was carried out. The
concentration of each metal of 50 mg/L was chosen because
it was indicated in previous literature to be the most favorable
in MFCs. For example, Li et al. (2019) investigated different
concentrations of Pb2+ and Cr6+ (50 ppm, 100 ppm, and 200
ppm) and found that 50 ppm provided the high removal effi-
ciency while having no toxic effect on the bacterial popula-
tion. Several other studies have concluded that a metal ion
concentration of 50 ppm on a laboratory scale has no toxic
effects (Choi et al. 2014; Li et al. 2008). Further, several stud-
ies already stated that MFCs are a successful approach to
decrease the metal concentration (Choi et al. 2014; Jia et al.
2013; Yaqoob et al. 2021b). For example, recently, Yaqoob
et al. (2021a) reported 60.33% (Pb2+) and 65.51% (Cd2+)
removal efficiencies within 40 days of benthic MFCs opera-
tion using the sweet potato waste as inoculation. Overall, in
Environ Sci Pollut Res
this study, 99.50% removal efficiency was achieved while
more than 90% Pb2+ and 82% Cd2+ were removed within 30
days. According to the observed trend, it was found that the
metal ion concentration decreases gradually. Initially, it was
not too high but with passage of time it boosted the removal
efficiency and achieved more than 90%. The high rate of
metal removal was achieved due to the fresh and stable inoc-
ulation source. The rotten rice is proved to be good a substrate
for microorganisms for their extracellular electron transporta-
tion. A complete trend of the present study is shown in
Table 3. Additionally, a comparative profile of the metal re-
moval via MFCs is compared in Table S1.
In MFCs, the bacterial species are involved in the generation
of electrons and protons which simultaneously reduces the sol-
uble states of metals into insoluble states. The produced biofilm
around the anode surface was scratched and operated with serial
dilution for bacterial isolation and identification process. Table 4
showed the identified bacterial species list on the surface of the
anode. These are responsible for electricity generation and metal
removal. According to the previous literature, it was found that
the identified bacterial species of the present study are well-
known exoelectrogens and metal-reducing species. For example,
Mathivanan et al. (2016) reported that the Lysinibacillus sp. is
the most effective species for the copper and cadmium ions
removal from wastewater. Similarly, Nandy et al. (2013) studied
the Lysinibacillus sp. as the most efficient exoelectrogens and
achieved the 85 mW/m2 power density using the Lysinibacillus
sp.. Further, Ayangbenro and Babalola (2020) observed that the
Bacillus species belong to the conductive pili-typed species and
are well-known metal-reducing species, particularly for
cadmium and lead ions. Later, Nimje et al. (2009) studied the
Bacillus species as an exoelectrogens species and achieved
0.000105 mW/m2 power density. Recently, some studies also
reported the Bacillus species and Lysinibacillus sp. as
exoelectrogens i.e., well-known metal-reducing species (Chuo
et al. 2020; Yaqoob et al. 2020d; Yaqoob et al. 2021a). The
chryseobacterium strains are well recognized in MFCs operation
as degrading the organic pollutant as reported by Nishioka et al.
(2016). However, according to the previous outcomes and pres-
ent results we can create a hypothesis that the present species are
capable of generating the energy as well as reducing the metal
ions from wastewater.
Biological tests
On the completion of the MFCs operation, SEM-EDX analy-
sis was carried out to evaluate the biological part of the oper-
ation. The anode and cathode electrodes were examined under
the SEM analysis before and after the operation. The SEM
images untreated anode and treated anode are shown in Fig.
3 (a-b). From the SEM images, it is seen that there are dense
bacterial species which is indicating that the operation did not
face any kind of toxicity. The dense and clear growth of
Environ Sci Pollut Res

Table 3 Removal trend of the metal via MFCs in the presence of rice rotten as inoculation source

Target Organic Incubation Concentration Time Removal Removal Removal Removal Removal Removal Removal
metals substrate source of each metal interval efficiency efficiency efficiency efficiency efficiency efficiency efficiency
ions (ppm) (Days) (%) of (%) of Pb2+ (%) of Cr3+ (%) of Ni2+ (%) of (%) of (%) of
Cd2+ Co2+ Ag+1 Cu2+

Cd2+ Rotten Synthetic 50 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Pb2+ rice waste- 10 43.20 37.35 57.60 52.75 66.98 52.50 45.00
Cr3+ water
20 74.70 65.40 76.10 78.10 82.00 88.00 74.90
Ni2+
Co2+ 30 82.2 95.71 96.18 89.50 89.82 99.50 99.88
Ag+1
Cu2+

bacterial species corresponding that the provided inoculation Recently, Yaqoob et al. (2021a) followed this concept to de-
source was sufficient to carry out the bacterial activities. In scribe the biological impact on the MFCs operation.
MFCs operation, the organic substrate plays a very important
role in bacterial growth and its stability. In the present study,
Discussion on electron transfer mechanism and metal
unique results were found. Similarly, the SEM images of cath-
removal
ode Fig. 3. (c-d) showed the dense bacterial species.
According to the SEM observation, it was observed that there
The MFCs approach mainly depends on the bacterial species
is almost similar morphology seen i.e., tube/rod-like filaments
activities. Several bacterial species are well-known
appendages. In the MFCs field, several studies reported that
exoelectrogens and metal-reducing were found in this study.
the filamentous appendages/rod-shaped morphology indicat-
In MFCs, the organic substrate is oxidized by bacterial species
ing the presence of conductive pili-based species such as
and generates the electron and protons (Nevin et al. 2009). In
Klebsiella pneumoniae, Lysinibacillus species,
the present work, bacterial species start the oxidation process
Acinetobacter species, Escherichia species, Bacillus species,
for rotten rice (rotten rice is a polysaccharide sugar, first it is
and Proteus species (Reddy et al. 2021; Yaqoob et al. 2020b).
converted into simple sugar which is further oxidized) to pro-
Further EDX was carried out at the end of operation to
duce the electrons and protons. The oxidation and reduction
examine the metal effect on the surface of the anode (biofilm).
reaction can be written as (Yaqoob et al. 2021c):
It was found that the metal concentration is successfully de-
→→Anodic reaction: Rotten rice C6H12O6 + 6H2O 6CO2
creased without affecting the biofilm growth. EDX spectra of
+ 24H+ +24e-
the treated anode are shown in Fig. 4. It was observed that
→Cathodic reaction: 24H+ + 24e- + 6O2 12H2O
mostly the percentage is carbon due to the graphite rod and
→Overall reaction: C6H12O6 + 6O2 6CO2 + 6H2O +
oxygen. There was no metal ion found on the surface of the
Energy + Biomass
anode. It means that there is no biosorption effect for metal
The produced electrons and protons are transferred to the
removal. This also indicates that the bacterial species showed
cathode from the anode. The proton can transfer directly from
good stability until the complete oxidation of the substrate.
anode to cathode while electrons are transferred via the outer

Table 4 The identified microbial species from anode biofilm of MFCs operation

Accession number1(6S rRNA Gene) Bacterial species Identity (%) Query cover (%)

NR_113722.1 Chryseobacterium gleum 95.51 99%

%
NR_042073.1 Lysinibacillus sphaericus 97.68% 98%
NR_041589.1 Lysinibacillus parviboronicapiens 95.65% 99%
NR_134074.1 Lysinibacillus varians 95.79% 98%
NR_114207.1 Lysinibacillus boronitolerans 95. 88% 98%
NR_042464.1 Chryseobacterium hispanicum 98% 88.91%
NR_159892.1 Chryseobacterium glaciei 98% 88.91%
NR_136869.1 Chryseobacterium hagamense 99% 91.49%
NR_115852.1 Bergeyella porcorum 99% 91.62%
NR_025569.1 Escherichia albertii 88% 82.32%
NR_126318.1 Cedecea lapagei 99% 80.71%
NR_118531.1 Siccibacter turicensis 92% 81.62%
NR_042648.1 Bacillus cecembensis 93.34% 98%

Fig. 3. a Untreated anode
electrode without biofilm b
treated anode electrode with
biofilm c Untreated cathode
electrode without biofilm d
treated cathode electrode with
biofilm
circuit. Additionally, before electron transportation toward the
cathode, there is a phase of electron transfer from bacteria to
the anode electrode. The produced biofilm round anode is a
collection of exoelectrogens of bacterial species. The domi-
nantly reported mechanism for electron transference from bac-
terial species to anode electrodes is shown in Fig. 5.
& Long-range electron transfer is belonging to conductive
pili-typed species. The conductive pili is a body part of
bacterial species which is conductive in nature as a metal.
This conductive part helps to transfer the electron directly
to another conductive anode very easily. Several conduc-
tive pili-typed species are well known in MFCs such as
G. sulfurreducens, Shewanella oneidensis, and
Pelotomaculum thermopropionicum (Yaqoob et al. 2020c).
& Short-range electron transfer is a mechanism in which
bacterial species utilized the oxidized shuttles and reduced
Fig. 4. EDX spectra of the treated anode electrode with biofilm on the completion of MFCs operation
Environ Sci Pollut Res
shuttles molecules. Some bacterial species are self-
producing the electron shuttles molecules to use further
to transfer the electrons. Desulfuromonadaceae and
Geobacteraceae are the most common examples. The
mainly stated self-electron shuttle molecules are OmcA,
MtrC, MtrF, and MtrE components.
& Electron transfer via using the redox-active proteins. The
Geobacter sulfurreducens is a commonly used mechanism
to transfer the electrons to the surface of the anode. Some
commonly reported redox-active proteins are OmcB,
OmcT, OmcS, OmcZ, and OmcE (Abbas et al. 2017).
Further, the biological test observations and literature indi-
cation proved that the present study follows the long-range
electron transfer mechanism via conductive pili. The identi-
fied bacterial species are also well-known exoelectrogens as
Environ Sci Pollut Res

Fig. 5. Mechanism for electron transfer from a bacterial cell to the surface of the anode

well as metal-reducing species in previous literature.

However, the soluble metal ions are converted into insoluble
states. Furthermore, AAS results only indicate the concentra- 3. Conversion of Cr3+ into Cr(s)
tions of metal ions that have been removed. In MFCs, the
metal ions that have been removed are converted to oxide
form and appear as sludge. The removed metal ions are effec-
tively converted to the oxide state, according to several re- →Cr3þ þ 3e− CrðsÞ
ports, and the resulting sludge contains the oxide form of →2Cr3þ þ 3H2 O Cr2 O3 þ 6Hþ
metals (Choi et al. 2014; Yaqoob et al. 2021a; Yaqoob et al.
2021b). The solid metal sludge is shown in Fig S1 (the image →Cr2 O3 þ 6e− þ 6Hþ 2CrðsÞ þ 3H2 O
was taken on the completion of the reaction). The final prod-
uct was like a solid sludge. Other side, the metal biochemical
reaction can be written as:
4. Conversion of Ni2+ into Ni (s)
1. Conversion of Cd2+ into Cd(s)

→Ni2þ þ 2e− NiðsÞ

→Cd2þ þ 2e− CdðsÞ →2Ni2þ þ 2H2 O 2NiO þ 4Hþ
→2Cd2þ þ 2H2 O 2CdO þ 4Hþ →NiO þ 2e− þ 2Hþ NiðsÞ þ H2 O
− þ
→CdO þ 2e þ 2H CdðsÞ þ H2 O

2. Conversion of Pb2+ into Pb(s) 5. Conversion of Co2+ into Co(s)

→Pb2þ þ 2e− PbðsÞ →Co2þ þ 2e− CoðsÞ

þ
→2Pb 2þ
þ 2H2 O 2PbO þ 4H →2Co2þ þ 2H2 O 2CoO þ 4Hþ
→PbO þ 2e− þ 2Hþ PbðsÞ þ H2 O →CoO þ 2e− þ 2Hþ CoðsÞ þ H2 O

6. Conversion of Ag 1+ into Ag(s)
→Ag1þ þ e− AgðsÞ
→2Ag1þ þ 2H2 O Ag2 O þ 4Hþ
→Ag2 O þ e− þ 2Hþ AgðsÞ þ H2 O
7. Conversion of Cu2+ into Cu(s)
→Cu2þ þ 2e− CuðsÞ
→2Cu 2þ
þ 2H2 O Cu2 O þ 4Hþ
→Cu2 O þ 2e− þ 2Hþ CuðsÞ þ H2 O
Effect of temperature and pH on MFCs operation
In MFCs operation generally, the most important parameters
are the temperature and pH of the operation. Several environ-
mental factors are involved in MFCs operation but these two
are the most significant. In the present study, selected temper-
ature ranges are 20 °C, 25 °C, 30 °C, and 35 °C. The reason
for the specific range is the MFCs operation cannot be oper-
ated at high temperature or low. Usually, the MFCs studies
were done within the range of 20 to 35 °C according to the
literature. However, the obtained results during the tempera-
ture optimization are shown in Fig. 6 (a-b). The maximum
achieved voltage was 41 mV at 30 °C while 40 mV at 25 °C
but the 20 °C offered lower voltage than others. The results
indicate that below 25 °C and above 35 °C the voltage gener-
ation trend is decreasing. Similarly, the highest removal effi-
ciency of metal ions was found at 30 °C. According to the
observation (Fig. 6b), below 20 °C or above 35 °C is not a
favorable condition for MFCs operation. Hsu et al. (2013) also
reported that the temperature of 30 °C is perfect for biofilm
growth and its stability.
Figure 6c shows the effect of pH on the voltage trend.
According to the voltage trend, it was found that an increasing
trend from 4 to until pH 7. After pH 8, the voltage trend was
decreased because bacterial species cannot survive in high
alkaline conditions. In the present study, it was found that
41 mV at pH 7. The pH 4 and 10 showed lower voltage
generation as compared to the neutral pH. Formerly, Huang
et al. (2012) studied the pH optimization and reported that the
pH 5.2 delivered low voltage with acidic sludge which sub-
stantially disrupted the voltage trend but later at pH 7 it was
regained. The unfavorable pH of the cell can disrupt the
Environ Sci Pollut Res
bacterial movements which led to a bad rate of voltage gener-
ation. Another side, the same pH was studied for the metal
removal. The maximum removal efficiency was found at pH 7
for all metal ions while pH 4 or 10 showed low removal
efficiency via MFCs. Therefore, the most suitable for MFCs
operation is at 7. At high alkaline or acidic conditions, special
care is needed to manage the bacterial activities. Several ear-
lier studies reported that pH 7 is suitable for voltage generation
and metal removal performance (Mohan et al. 2011; Mohan
et al. 2009; Yuan et al. 2011). Recently Yaqoob et al. (2021a)
reported the effect of the pH on energy generation and metal
removal and they claimed that pH 7 is perfect. Despite all, the
present observation in terms of energy generation and removal
is found higher than their reported results. It means that the use
of inoculum source also contributes to the performance at pH
7 at room temperature.
Conclusion and future recommendations
The efficiency of rotten rice as a substrate in MFCs to generate
energy and decrease metal ions concentration from synthetic
wastewater was highlighted in this study. In comparison to
many previous studies, the waste-derived substrate in MFCs
delivered unique results. The 510 mV (0.5V) voltage genera-
tion was achieved within 14 days. The maximum removal
efficiency is 99.88% for Cu2+ within 30 days. While other
toxic metals showed more than 80% removal efficiency. In
the present study, the biological characterizations revealed
that the produced biofilm activities were stable and good.
The identified bacterial species also indicate a good sign for
effective removal of toxic metals. Furthermore, the MFCs
were found to be best operated at room temperature and neu-
tral pH conditions in a multi-parameter optimization study.
Despite all, there are still some challenges that are hindering
the performance of MFCs for practical applications at an in-
dustrial scale. The most critical challenge in MFCs is the low
transportation of electrons from anode to cathode. Although
the percentages of metal removal are acceptable in the current
condition, there are still questions on preserving the energy.
The most important factor in MFCs is the electrode material
for efficient electron transportation and a stable substrate.
Currently, no material can provide highly efficient electron
transportation in the application of MFCs. Several efforts are
progressing to develop an efficient material for electrodes.
Nowadays, waste-derived graphene derivatives and their com-
posites with metal oxide showed the potential to revolutionize
electron transport. The utilization of waste to developing the
material for electrodes can be a long-lasting survival solution
for MFCs. Further, in the future, the utilization of waste as a
substrate such as rotten rice, sugar cane waste, carbohydrate-
based waste, etc. can be an alternative source to improve shelf-
life of the substrate in MFCs.
Environ Sci Pollut Res
Fig. 6. Effect of temperature on a voltage generation b removal efficiency and effect of pH on c voltage generation d removal efficiency
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s11356-021-15104-w.
Acknowledgements This research article was financially supported by
Universiti Sains Malaysia, (Malaysia) under the Research Grant; 304/
PKIMIA/6501153/E128.
Data availability statement The authors confirm that all data underlying
the findings are fully available without restriction. Data can be obtained
after submitting a request to the corresponding/first author.
Author contribution Mohamad Nasir Mohamad Ibrahim, Asim Ali
Yaqoob: Conceptualization. Najwa Najihah Mohamad Daud, Asim Ali
Yaqoob: Methodology, writing-original draft preparation, visualization,
electromicrobiology investigation. Akil Ahmad: English editing and re-
vision of the manuscript. Mohamad Nasir Mohamad Ibrahim:
Supervision, funding acquisition. This article has been read and approved
by all listed authors.
Declarations
Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable.
Competing interests The authors declare no competing interests.
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