Science of the Total Environment 686 (2019) 538–545

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Increasing of electricity production from Echinodosus

cordifolius-microbial fuel cell by inoculating Bacillus thuringiensis
Chairat Treesubsuntorn a,⁎, Wachira Chaiworn a, Werasak Surareungchai b, Paitip Thiravetyan b
a
Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkok 10150, Thailand
b
School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10150, Thailand

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Inoculation of Bacillus thuringiensis to

wetland-microbial fuel cell can promote
electric power generation.
• Nitrate and PO3− 4 are the major anion
driving electric current in the system.
• Echinodosus cordifolius maintains Bacil-
lus thuringiensis density for long term
operation.
• Wetland-microbial fuel cell inoculated
with Bacillus thuringiensis produces
20–35 mW m−2 maximum power den-
sity.
• The system can self-sustain for longer
than 180 days.

a r t i c l e i n f o a b s t r a c t

Article history: The wetland-microbial fuel cell (MFC) is a novel electricity generating technology. However, these systems can
Received 22 April 2019 generate only limited electric energy. Since nitrification is a key mechanism driving electrical power in
Received in revised form 24 May 2019 wetland-MFC systems, an effective nitrifying bacteria, Bacillus thuringiensis, was used to inoculate a wetland-
Accepted 4 June 2019
MFC to enhance the maximum power density of the system. B. thuringiensis effectively enhanced the maximum
Available online 05 June 2019
power density, producing about 20–35 mW m−2 of maximum power density. Interestingly, over the first 120 days
Editor: Frederic Coulon of operation, the wetland-MFC system with only B. thuringiensis generated more power than a system containing
an Echinodosus cordifolius plant in addition to B. thuringiensis, because E. cordifolius can took up nitrate (NO−3) and
Keywords: phosphate (PO3− 3−
4 ) in system's solution. Nitrate and PO4 act as important anions driving electric current in the
Bacillus thuringiensis system. After 120 days of operation though, the combined E. cordifolius and B. thuringiensis system maintained
Echinodosus cordifolius 20–35 mW m−2 maximum power density and the maximum power density of the system only inoculated with
Electrical power B. thuringiensis decreased continuously. Gene (16S rRNA) copy numbers for B. thuringiensis showed that when
Nitrification E. cordifolius was presented, the bacterium was able to continue growing after 120 days of operation.
Wetland-MFC
B. thuringiensis did not grow as well after 120 days in the system that did not contain a plant. This study presents
a strategy for enhancing electric power output from a wetland-MFC by inoculating the system with
B. thuringiensis and maintaining the bacterium's population with the support of an E. cordifolius plant. The result
clearly show that B. thuringiensis can enhance electric power generation in the presence of the plant and the sys-
tem can self-sustain for longer than 180 days of operation while producing 20–35 mW m−2 maximum power
density.
© 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: King Mongkut's University of Technology Thonburi, Pilot Plant Development and Training Institute, 49 Soi Thian Thale 25, Bang Khun Thian Chai Thale Road,
Tha Kham, Bang Khun Thian, Bangkok 10150, Thailand.
E-mail address: chairat.tre@kmutt.ac.th (C. Treesubsuntorn).

https://doi.org/10.1016/j.scitotenv.2019.06.063
0048-9697/© 2019 Elsevier B.V. All rights reserved.
 C. Treesubsuntorn et al. / Science of the Total Environment 686 (2019) 538–545
1. Introduction
Energy supply is one of the major 21st century challenges (Saba
et al., 2019). Research groups around the world have studied and ap-
plied several clean technologies to generate electrical energy (Pérez-
Lombard et al., 2008; Kothari et al., 2011). The microbial fuel cell
(MFC) was proposed as a sustainable and valuable technology that
uses bioelectrochemistry to generate electricity (Wang and Ren, 2013;
Li et al., 2013; Xu et al., 2016). However, limited carbon sources for bac-
terial growth can limit MFC electricity production. MFC systems require
constant bacterial nutrient supplies to maintain bacterium density and
electrical production, and resulting in high MFC operational costs. The
mutualistic symbiosis among plants and microorganisms are well
known (Afzal et al., 2014; Kobayashi and Palumbo, 2000; Rajkumar
et al., 2009). Plants fix CO2 through photosynthesis to generate carbohy-
drates that accumulate in plant cells where they are used for growth,
metabolism, and storage. In addition, some organic compounds pro-
duced by plant are released from the root to the environment
(Gregory, 2008). Soil microorganisms use these root exudate com-
pounds as a carbon source (Strik et al., 2008). Therefore, combining
wetland conditions and a MFC might enhance MFC electricity produc-
tion (Türker and Yakar, 2017; Nitisoravut and Regmi, 2017; Lu et al.,
2015), improve the phytoremediation efficiency of wastewater
(Corbella and Puigagut, 2018), and reduce wetland greenhouse gas
emissions (Wang et al., 2019). In such a system, plants would provide
bacteria with nutrients and the bacteria would generate electricity.
Wetland-MFC investigations have received worldwide interest (Yadav
et al., 2012; Doherty et al., 2015; Corbella et al., 2015; Fang et al.,
2015; Srivastava et al., 2017).
During nitrification ammonium (NH4) is transformed to nitrate
(NO−3), and in an MFC a proton from the anode is moved from the bot-
tom of the system (i.e., anaerobic bacterial zone) to the top of the sys-
tem (i.e., aerobic bacterial zone) (Xu et al., 2018a, b). This behaviour
generates a positive charge on the top of system and a difference in
the total charge between the top and bottom of the system, thus forcing
electron movement and generating electricity (Logan, 2009; Kim and
Logan, 2013). Given the oxygen concentration gradient in the system,
growth of suitable microorganism species in the cathode and anode
sides of a MFC can enhance power generation (Xu et al., 2019). There-
fore, inoculating the wetland-MFC with nitrifying bacteria is an effective
method to enhance electricity generation. Bacillus thuringiensis is an
aerobic bacterium that exhibits potential to provide nitrification ser-
vices (Santawee et al., 2019). B. thuringiensis can transform
trimethylamine (TMA) to NO−3 by nitrification and transform NO−3 to ni-
trogen by denitrification (Siswanto and Thiravetyan, 2016). This bacte-
ria is also an endophyte that can non-pathogenically colonize plant
roots (Siswanto and Thiravetyan, 2016). Inoculating the water surface
of the wetland-MFC (aerobic zone) with B. thuringiensis might improve
electric production.
Echinodosus cordifolius is a wetland plant used effectively for
phytoremediation (Prum et al., 2018; Teamkao and Thiravetyan,
2015). This plant can absorb pollutants in contaminated wastewater,
is easy to grow, and is tolerant to a range of environmental conditions
(Sriprapat and Thiravetyan, 2011; Sriprapat et al., 2011; Noonpui and
Thiravetyan, 2011). Echinodosus cordifolius also releases oligo- and
mono-saccharides and minerals into the rhizosphere (Torit et al.,
2012). Therefore, E. cordifolius was selected as an ideal species for fur-
ther investigation. Herein, wetland-MFCs containing E. cordifolius as
the primary plant species were inoculated with B. thuringiensis and in-
vestigated for their capacity to improve electricity production. Nega-
tively charged compounds and bacterial densities, were investigated.
Electricity production, maximum power density, and suitable resistance
539
for maximum power density were also analysed. This study confirms
the proposition that inoculating a wetland-MFC with an effective micro-
organism enhances electricity production. The results will benefit con-
tinued development of wetland-MFC.
2. Materials and methods
2.1. Plant and bacteria preparation
E. cordifolius was purchased from a local market in Thailand and
grown in a greenhouse at 35–40 °C and 60–70% humidity at the Reme-
diation Laboratory of King Mongkut's University of Technology
Thonburi, Bangkhuntien Campus. The plants were around 3 months
old and homogenously sized plants (1000–1200 cm2 of leaf area)
were chosen for each condition and replicate. Plants were cultivated in-
dividually in pots containing clay soil (40–50% clay, 25–30% silt, and
25–30% sand) with a pH of 7.10, organic matter content of 0.64, and cat-
ion exchange capacity (CEC) around 23.4 cmol kg−1. Fertilizer (N-P-K =
16–20-0) was supplied once after 30 days growth.
The B. thuringiensis (CP010577.1) bacteria was taken from a culture
maintained at the Remediation Laboratory. The bacteria was grown in
nutrient broth (Himedia, USA) containing 5.0 g peptone, 5.0 g sodium
chloride, 1.5 g HM peptone B#, and 1.5 g yeast extract.
2.2. Wetland-MFC setup and operation
The wetland-MFC system was made from polyvinyl chloride plastic
pipe with a 15 cm diameter. The system can be separated into 4 parts:
1) plant growth section 2) anode section 3) bottom lid and 4) joint
(Fig. 1). Plant growth section and anode section was connected by
joint, which have a hole in the centre for water contact between
anode and cathode. The plastic pipe height was 45 cm long in total
and 40 cm long in the plant growth section (8 L of total volume; 6.2 L
of plant growth section volume) and filled with 2 L of deionized (DI)
water and 4 kg of the clay soil (40–50% clay, 25–30% silt, and 25–30%
sand) with a pH of 7.10, organic matter content of 0.64, and cation ex-
change capacity (CEC) around 23.4 cmol kg−1 as same as in
Section 2.1. All electrochemical system was placed in the system. Car-
bon fibre (hydrate carbon) sheet, width 15 cm with length 4 cm and
1 mm thickness, was used as a cathode and anode. The carbon fibre
plate of the anode was located in the container at the bottom of the sys-
tem (35 cm below system soil surface), while the carbon fibre plate for
the cathode was installed under the surface water at the top of the sys-
tem. Copper electric wire was used to connect the cathode and anode.
Since water can be evaluated and take up by plant, the level of water
was controlled by supplying DI water (350 mL) every day to maintain
the system water level (5 cm above soil surface). One E. cordifolius
plant with 1000–1200 cm3 of leaf area was added to each pot system.
A multimeter was used to measure electricity production. These
wetland-MFC systems were placed in a controlled room at 30–35 °C,
60–70% humidity, and a photon flux density (light intensity) of 100
μmol m−2 s−1. The treatments consisted of three separated conditions:
A) a wetland-MFC system with an E. cordifolius plant (Soil
E. cordifolius), B) a wetland-MFC system with an E. cordifolius plant
and inoculated with B. thuringiensis (Soil E. cordifolius + B.
thuringiensis), and C) a wetland-MFC system with B. thuringiensis and
no E. cordifolius plant (Soil + B. thuringiensis) when control systems
was D) a wetland-MFC system filled with only soil and operated with-
out an E. cordifolius plant (Soil). All treatments and the control were
set up in triplicate.
540 C. Treesubsuntorn et al. / Science of the Total Environment 686 (2019) 538–545

Fig. 1. A schematic diagram of the wetland-MFC system (A) and a photograph of the wetland-MFC system (B).

Fig. 2. Wetland-MFC system polarization curves representing the relationship between power (mW) and current (mA) under various external resistances obtained on (A) day 2, (B) day
30, (C) day 60, (D) day 120, (E) day 150, and (F) day 180.
 C. Treesubsuntorn et al. / Science of the Total Environment 686 (2019) 538–545 541

2.3. Polarization studies 3. Results and discussion

To quantify polarization the carbon fibre plates of cathode and anode
were connected with copper wire and the end of the wire was allowed
to extend out into the wetland-MFC system. Two digital multimeter (a
DT830B, China; and a Yugo Digital MultiMeter M920R) were installed
to measure current and voltage, respectively. The output voltage was
manually recorded every day for first 30 day in order to make sure sta-
bility of output voltage and every week during day 30–180. Since the
polarization curves from each wetland-MFC system were related to ex-
ternal electrical resistance, the maximum power was determined by po-
larization curves. In this experiment, external resistance was varied
every 20 min from 200,000 to 10 Ω periodically. Each measurement
had an equilibration time of 10 min. Polarization measurements were
performed in triplicate.
2.4. Analysis of negatively charged compounds
Negatively charged compounds, including NO−3 and phosphate
(PO3−
4 ), were quantified with ion chromatography (IC) (Dionex
Integrion RFIC HPIC System, Thermo Fisher, USA) using a Dionex
anion self-regenerating suppressor ASRS 300 2 mm. Dionex EGC 500
potassium hydroxide (KOH) REIC™ and HPIC™ was used as an eluent
generator cartridge. Dionex IonPac™ AG17-C 4 × 50 mm and 4
× 250 mm columns were used.
2.5. B. thuringiensis quantification
3.1. Increased electricity production from a wetland-MFC system with
B. thuringiensis inoculation
Wetland-MFC systems inoculated with 2% B. thuringiensis
(109 cfu mL−1) had greater electricity productivity compared to
wetland-MFC systems without B. thuringiensis. External loads were the
most important factor for power generation (Wang et al., 2018). The
electric current of the wetland-MFC system under various external
loads (10–200,000 Ω) was measured and the relationship between cur-
rent and power of the wetland-MFC systems was analysed. Wetland-
MFC system polarization curves indicated the maximum power density
of each condition (Fig. 2). The external resistance at which the system
generated the greatest power was around 300–2000 Ω, and the electric
power generated by the wetland-MFC system was around 0.05–0.5 mW
until 120 days. The wetland-MFC systems (Soil E. cordifolius + B.
thuringiensis) and (Soil B. thuringiensis) produced greater maximum
power than the other wetland-MFC systems (Soil and Soil
E. cordifolius). In addition, Soil + B. thuringiensis produced more electric-
ity than Soil E. cordifolius B. thuringiensis (Fig. 2A–D.). Soil + B.
thuringiensis generated around 0.13–0.6 mW per system, while Soil
E. cordifolius + B. thuringiensis generated 0.13–0.3 mW per system. In-
terestingly, after the wetland-MFC systems were operated for
150 days, the wetland-MFC system without E. cordifolius (i.e., Soil
B. thuringiensis) produced less electricity than the Soil E. cordifolius
+ B. thuringiensis treatment (Fig. 2E–F.). Soil B. thuringiensis generated
around 0.13–0.35 mW per system, while Soil E. cordifolius + B.

Soil samples were taken from each treatment after 60, 90, and
120 days of operation and crushed in a mortar and pestle with liquid ni-
trogen. The FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA)
were used to extract DNA from crushed samples following the manufac-
turer's instruction. DNA integrity was examined using a Nanodrop spec-
trophotometer (Thermo Fisher Scientific, USA). An Agilent Mx3005P
qPCR cycler (Santa Clara, CA, USA) was used to perform quantitative
PCR (qPCR). Each 20 μL qPCR reaction contained 1 μL of template DNA
(20 ng), 0.4 μL of 50 x ROX Low (KAPA BIOSYSTEMS, Wilmington, MA,
USA), 10 μL of 2 x KAPA SYBR FAST qPCR Master Mix Universal (KAPA
BIOSYSTEMS), and 0.4 μL of each primer (200 nM) (in triplicate). The
qPCR program consisted of an initial denaturation (95 °C) for 10 min;
40 cycles of denaturation (95 °C) for 30 s, annealing (60 °C) for 10 s,
and extension for 20 s at 72 °C; one final cycle of 95 °C for 1 min, 55
°C for 30 s, and 95 °C for 30 s (Khaksar et al., 2017). The
B. thuringiensis specific primers used were 5′-CCAGCAGCCGCGGTAA
TACG-3′ forward primer and 5′-TACCAGGGTATCTAATCC-3′ reverse
primer.

2.6. Statistical analysis

All the experiments were done in triplicate. The average and stan-
dard deviation (SD) of all variables were calculated. For the concentra-
tions of negatively charged compounds, including NO−3 and PO3− 4 ,
means and SD were presented in a bar graph and one-way ANOVA
was used to compare means among treatments. Additionally, Duncan's
multiple range tests at the 95% confidence level were applied for group
classification. Bacterial copy numbers were presented as a mean and SD
in a bar graph and a one-way ANOVA was used to compare mean of
each treatment and a Duncan's multiple range test at the 95% confi-
dence level was applied for group classification. All statistical analysis
in this study were performed in SPSS version 20.
Fig. 3. Internal resistance (Ω) in the wetland-MFC systems 0–30 day (daily record)
(A) and0–180 day (weekly record) (B) calculated from the slope of the I-V curve to
various external resistances. Measured from day 0 to 180.
542 C. Treesubsuntorn et al. / Science of the Total Environment 686 (2019) 538–545

thuringiensis generated 0.35–0.45 mW per system and maintained max-
imum power output for more than 180 days.
The wetland-MFC system containing only soil had very high internal
resistance, around 5000 Ω and tended to increase after 120 days of op-
eration. This resulted to limit electricity generation. In this case, limit of
bacteria carbon sources might inhibit the growth of bacteria, which is
the key organism to drive electricity in the system since microorganism
is required to oxide the organic matter in order to release electron and
proton for electricity generation. The internal resistance in the other
wetland-MFC systems was less than 2000 Ω (Fig. 3A–B.). In this case,
the stability of internal resistance can be found in daily internal resis-
tance record for the first 30 day operation (Fig. 3A.). The result can be
explained by secretion of root exudate from E. cordifolius, which bacteria
can utilized as a carbon sources for survive. Oxidation of root exudate
organic matter can increase electron and proton in the system, result
to decrease system internal resistant and increase electric generation.
For Soil + B. thuringiensis condition, although soil carbon sources for
bacteria growth was limited, B. thuringiensis might high enough poten-
tial to grown and provide nitrification process for 120 day but not for
150 day.
The stability of power densities can be found in daily power densities
record for the first 30 day operation (Fig. 4A.). After 30 day operation,
power densities were recorded weekly. The result showed that the
power densities of wetland-MFC systems were around 5–35 mW m−2
(Fig. 4B). The wetland-MFC system with B. thuringiensis (Soil
B. thuringiensis) generated the greatest power density up to 120 days
of operation (20–35 mW m−2). The complete wetland-MFC system
(Soil E. cordifolius + B. thuringiensis) generated greater power density,
around 25–35 mW m−2, from day 120 to 180 days of operation. The
other wetland-MFC systems (Soil E. cordifolius and Soil) only generated
less than 10 mW m−2.
In this study, B. thuringiensis seemed to increase the water NO−3 con-
centration. While other microorganisms from clay soil oxidize organics
matter and release electron and proton (H ). Elevated NO−3 in solution is
an important factor for electricity generation (Xu et al., 2018a, 2018b). A
high NO−3 concentration at the water surface of the wetland-MFC system
(cathode) induced H movement from the bottom of the system (anode)
to the cathode.
The accumulation of H at the cathode can generate the higher volt-
ages necessary to drive the movement of electrons from the anode to

Fig. 4. Maximum power densities (mW m−2) of the wetland-MFC systems 0–30 day (daily record) (A) and 0–180 day (weekly record) (B), which were derived from the relationship
between power (mW) and current (mA) under various external resistances using the Wetland-MFC system polarization curves.
 C. Treesubsuntorn et al. / Science of the Total Environment 686 (2019) 538–545
the cathode through the electric wire (Logan, 2009; Kim and Logan,
2013). Although the complete wetland-MFC system (Soil
E. cordifolius B. thuringiensis) was also inoculated with B. thuringiensis,
it still produced less electrical power than wetland-MFC system with
only B. thuringiensis (Soil B. thuringiensis) for the first 120 days of oper-
−
ation. Since plants readily uptake PO3− 4 (Torit et al., 2012) and NO3, the
E. cordifolius plants likely reduced the anion concentration in the
water, and thus, reduced the power output of the wetland-MFC systems
containing them. After the plants took up the anions, power density was
significantly decreased. However, with long-term operation (after
120 days), the complete wetland-MFC system (Soil E. cordifolius
B. thuringiensis) was better able to maintain the microorganisms in the
system, which resulted in greater maintenance of electrical power in
the system, while the system with only B. thuringiensis (Soil
B. thuringiensis) decreased after 120 days operation. An equivalent re-
sult was found with another wetland-MFC system operated over the
long-term with mixed microorganisms. Wang et al. (2017) proposed
that the plant is the key to sustainable energy production in wetland-
MFC systems.
The complete wetland-MFC system (Soil E. cordifolius + B.
thuringiensis) resulted in a potential production of 20–35 mW m−2
with long-term operation. This power density could provide an alterna-
tive energy source within an ecologically sustainable system. In a previ-
ous study Canna indica was used in a wetland-MFC system and
generated about 31.04 mW m−2 (Srivastava et al., 2015), while a
wetland-MFC system with Juncus effusus only generated a maximum
power density around 21 mW m−2 (Wang et al., 2017).
In some cases wetland-MFC systems with suitable designs can gen-
erate more electric power, ranging 20–120 mW m−2 (Saba et al., 2015;
Wu et al., 2016; Corbella et al., 2016; Türker and Yakar, 2017). For exam-
ple, an up flow wetland-MFC system with Elodea nuttallii and activated
sludge generated 185 mW m−3 (Oon et al., 2017). Numerous studies
promote the potential large-scale applicability of this technology.
Fig. 5. NO−3 and PO3−
4 concentrations (mg L ) in the wetland-MFC systems from day 0 to day180.
543
3.2. Effect of water soluble NO−3 and PO3−
4 on electricity productivity
Based on the electricity production, inoculating wetland-MFC sys-
tems with B. thuringiensis promotes electrical power generation. Since
B. thuringiensis has the potential increase NO−3 concentrations in the
water (Santawee et al., 2019; Siswanto and Thiravetyan, 2016), the
mechanism enhancing electrical power of wetland-MFC systems was
potentially mediated through NO−3. Therefore, the NO−3 concentration
in the surface water was measured (Fig. 5). The initial (day 0) NO−3 con-
centrations were very low. After 30–60 days of operation the NO−3 con-
centrations in the complete wetland-MFC system (Soil E. cordifolius
+ B. thuringiensis) and inoculated system (Soil B. thuringiensis) had sig-
nificantly higher NO−3 concentrations relative to the non-inoculated
wetland-MFC system (Soil). Interestingly, Soil B. thuringiensis had a sig-
nificantly greater NO−3 concentration than Soil E. cordifolius + B.
thuringiensis at day 90, likely because E. cordifolius can take up large
quantities of NO−3 from the system solution. These results are consistent
with the maximum power output since the inoculated wetland-MFC
system (Soil B. thuringiensis) generated more electricity than the com-
plete wetland-MFC system (Soil E. cordifolius + B. thuringiensis). How-
ever, the complete wetland-MFC system (Soil E. cordifolius + B.
thuringiensis) did maintain the NO−3 concentration in the system from
90 to 120 days of operation. In contrast, the NO−3 concentration in the in-
oculated wetland-MFC system (Soil B. thuringiensis) decreased after
120 days of operation. The non-inoculated wetland-MFC system (Soil
E. cordifolius) and control (Soil) had very low NO−3 concentrations
(0–0.1 mg L−1).
The inoculated wetland-MFC system (Soil B. thuringiensis) had a
high PO3−4 concentration compared to the other treatments during the
60–120 day period. However, the Soil E. cordifolius + B. thuringiensis
treatment had higher PO3− 4 concentrations than Soil B. thuringiensis at
150–180 days of operation (Fig. 5) possibly because E. cordifolius took
up PO3−4 in the early operation period from 0 to 120 days and released
PO3−
4 after 120 days of operation. A previous study using E. cordifolius
suggested that it releases PO3−
4 to the water (Torit et al., 2012). These re-
sults were also consistent with the electric power generation results,
−1
544 C. Treesubsuntorn et al. / Science of the Total Environment 686 (2019) 538–545
Fig. 6. Bacillus thuringiensis 16S rRNA gene copy number per g of cathode soil in each treatment after 0, 90, and 180 days of operation.
suggesting that the NO−3 and PO3−
4 concentrations in wetland-MFC sys-
tems are key factor effecting electricity production. A similar result
was described with a system composed of multiple bio-cathodes in a
constructed wetland-microbial fuel cell used to generate electric
power. The relationship between power production and the systematic
nitrification rate was clearly presented (Xu et al., 2016). Wang et al.
(2017) also found that the denitrification process was accelerated in a
constructed wetland-microbial fuel cell system.
3.3. Echinodosus cordifolius promoted B. thuringiensis growth for long-
term operation
NO−3 and PO3− 4 concentrations are likely key factors driving electricity
production in wetland-MFC systems. B. thuringiensis also showed po-
tential to enhance electric power generation, nitrification, and PO3− 4 cathode and anode would be recommended to study. In addition, a ca-
solubilisation in wetland-MFC systems. Therefore, this bacterium was
the main organism driving electric current in the wetland-MFC system.
The number of B. thuringiensis in the wetland-MFC systems was also po-
tentially an important parameter (Fig. 6.). At day 0 a subsample of initial
soil (to 5 cm depth in cathode) was taken to quantify B. thuringiensis 16S
rRNA gene copy number using strain specific primers. B. thuringiensis
was not found in the initial (non-inoculated) soil. After 90 days of oper-
ation, the top 5 cm of the cathode soil was again subsampled to analyse
B. thuringiensis 16S rRNA gene copy number. At 90 days the complete
wetland-MFC system (Soil E. cordifolius + B. thuringiensis) and inocu-
lated (Soil B. thuringiensis) treatment had significantly higher
B. thuringiensis 16S rRNA gene copy number relative to the other treat-
ments. However, after 180 days of operation only the complete
wetland-MFC system (Soil E. cordifolius + B. thuringiensis) had a higher
B. thuringiensis 16S rRNA gene copy number than other treatments. Al-
though B. thuringiensis is an important microorganism that enhanced
electricity generation in the system, it may not be able to persist without
E. cordifolius. In this system, B. thuringiensis may have required carbon
sourced from E. cordifolius. The carbon sources B. thuringiensis was uti-
lizing may have become limiting in the soil after 120 days of operation
without E. cordifolius. E. cordifolius likely provided nutrients for the bac-
teria to grow (Siswanto and Thiravetyan, 2016). The result can be used
to explain power production decreasing in (Soil B. thuringiensis) treat-
ment in Section 3.1. The anode soil was sampled at 30 cm depth and also
analysed for B. thuringiensis 16S rRNA gene copy number.
B. thuringiensis was not found in the anode of any treatments. Since
B. thuringiensis is an aerobic bacterium, isolated from a fish shop in
this case (Siswanto and Thiravetyan, 2016), it was capable of growing
in the wetland-MFC system's surface soil that had adequate oxygen
for its growth, but not in the anoxic deep soil. These results suggest
that the difference in chemical and microbial composition between
the cathode and anode determine electric power generation (Guan
et al., 2019).
4. Conclusion
An E. cordifolius wetland-MFC system inoculated with Bacillus
thuringiensis can produce 20–35 mW m−2 maximum power density
and maintain operation for 180 days. This technology is ecologically
friendly and available for large-scale application. If wetland-MFC sys-
tems are properly designed with effective bacterial inoculation, the sys-
tems can generate ample electric power. Further studies will investigate
this B. thuringiensis inoculated E. cordifolius wetland-MFC system at a
pilot scale and with field trials. In order to clarify the mechanism of
this developed system, the amount of the microorganisms in the system
pacitor for low electrical power will require development in order to
collect the power and stabilize electric support. This development
holds potential to initiate the creation of innovative applications, for ex-
ample a wetland-MFC system combined with a streetlight. The plant-
bacteria interactions in these wetland-MFC systems that allow mainte-
nance of bacterial population densities and electricity generation are
also interesting to investigate further.
Acknowledgements
This work was financially supported by King Mongkut's University of
Technology Thonburi (KMUTT) through the “KMUTT 55th Anniversary
commemorative fund”.
References
Afzal, M., Khan, Q.M., Sessitsch, A., 2014. Endophytic bacteria: prospects and applications
for the phytoremediation of organic pollutants. Chemosphere 117, 232–242.
Corbella, C., Puigagut, J., 2018. Improving domestic wastewater treatment efficiency with
constructed wetland microbial fuel cells: influence of anode material and external re-
sistance. Sci. Total Environ. 631-632, 1406–1414.
Corbella, C., Guivernau, M., Vinas, M., Puigagut, J., 2015. Operational, design and microbial
aspects related to power production with microbial fuel cells implemented in con-
structed wetlands. Water Res. 84, 232–242.
Corbella, C., Garfí, M., Puigagut, J., 2016. Long-term assessment of best cathode position to
maximise microbial fuel cell performance in horizontal subsurface flow constructed
wetlands. Sci. Total Environ. 5634, 48–455.
Doherty, L., Zhao, Y., Zhao, X., Hu, Y., Hao, X., Xu, L., Liu, R., 2015. A review of a recently
emerged technology: constructed wetland – microbial fuel cells. Water Res. 85,
38–45.
Fang, Z., Song, H.L., Cang, N., Li, X.N., 2015. Electricity production from Azo dye wastewa-
ter using a microbial fuel cell coupled constructed wetland operating under different
operating conditions. Biosens. Bioelectron. 68, 135–141.
Gregory, P.J., 2008. The Rhizosphere, Plant Roots: Growth, Activity and Interactions With
the Soil. John Wiley & Sons, pp. 216–252.
 C. Treesubsuntorn et al. / Science of the Total Environment 686 (2019) 538–545
Guan, C.Y., Hu, A., Yu, C.P., 2019. Stratified chemical and microbial characteristics between
anode and cathode after long-term operation of plant microbial fuel cells for remedi-
ation of metal contaminated soils. Sci. Total Environ. 670, 585–594.
Khaksar, G., Treesubsuntorn, C., Thiravetyan, P., 2017. Impact of endophytic colonization
patterns on Zamioculcas zamiifolia stress response and in regulating ROS, tryptophan
and IAA levels under airborne formaldehyde and formaldehyde-contaminated soil
conditions. Plant Physiol. Biochem. 114, 1–9.
Kim, Y., Logan, B.E., 2013. Microbial desalination cells for energy production and desalina-
tion. Desalination 308, 122–130.
Kobayashi, D.Y., Palumbo, J.D., 2000. Bacterial endophytes and their effects on plants and
uses in agriculture. In: Bacon, C.W., White, J.F. (Eds.), Microbial Endophytes. Marcel
Dekker, Inc., New York, N.Y, pp. 199–233.
Kothari, D.P., Singal, C.K., Ranjan, R., 2011. Renewable Energy Sources and Emerging Tech-
nologies. PHI Learning Pvt. Ltd.
Li, W.W., Yu, H.Q., He, Z., 2013. Towards sustainable wastewater treatment by using mi-
crobial fuel cells-centered technologies. Energy Environ. Sci. 7, 911–924.
Logan, B.E., 2009. Exoelectrogenic bacteria that power microbial fuel cells. Nat. Rev.
Microbiol. 7, 375–381.
Lu, L., Xing, D., Ren, Z.J., 2015. Microbial community structure accompanied with electric-
ity production in a constructed wetland plant microbial fuel cell. Bioresour. Technol.
195, 115–121.
Nitisoravut, R., Regmi, R., 2017. Plant microbial fuel cells: a promising biosystems engi-
neering. Renew. Sust. Energ. Rev. 76, 81–89.
Noonpui, P., Thiravetyan, P., 2011. Treatment of reactive azo dye from wastewater by
burhead (Echinodorus cordifolius L.) in constructed wetland: effect of molecular
size. J. Environ. Sci. Health A 46, 709–714.
Oon, Y.L., Ong, S.A.L., Ho, N., Wong, Y.S., Dahalan, F.A., Oon, Y.S., Nordin, N., 2017. Role of
macrophyte and effect of supplementary aeration in up-flow constructed wetland
microbial fuel cell for simultaneous wastewater treatment and energy recovery.
Bioresour. Technol. 224, 265–275.
Pérez-Lombard, L., Ortiz, J., Pout, C., 2008. A review on buildings energy consumption in-
formation. Energy Build. 40, 394–398.
Prum, C., Dolphen, R., Thiravetyan, P., 2018. Enhancing arsenic removal from arsenic-
contaminated water by Echinodorus cordifolius? Endophytic Arthrobacter
creatinolyticus. J. Environ. Manag. 213, 11–19.
Rajkumar, M., Ae, N., Freitas, H., 2009. Endophytic bacteria and their potential to enhance
heavy metal phytoextraction. Chemosphere 77, 153–160.
Saba, B., Jabeen, M., Aziz, I., Khalid, A., Christy, A.D., 2015. Role of agro-waste based acti-
vated biochar and microbes in reactive black dye degradation from microcosm con-
structed wetlands. Int. J. Phytoremediation 7, 1060–1067.
Saba, B., Khan, M., Christy, A.D., Kjellerup, B.V., 2019. Microbial phyto-power systems – a
sustainable integration of phytoremediation and microbial fuel cells.
Bioelectrochemistry 127, 1–11.
Santawee, N., Treesubsuntorn, C., Thiravetyan, P., 2019. Using modified coir pith–glucose
syrup beads inoculated with Bacillus thuringiensis as a packing material in
trimethylamine (fishy odor) biofilter. Atmos. Pollut. Res. (In press). https://www.
sciencedirect.com/science/article/abs/pii/S1309104218307037#undfig1.
Siswanto, D., Thiravetyan, P., 2016. Improvement of trimethylamine uptake by Euphorbia
milii: effect of inoculated Bacteria. JTLS 6, 123–130.
Sriprapat, W., Thiravetyan, P., 2011. Phytoremediation of diethylene glycol contaminated
wastewater by Echinodorus cordifolius. Int. J. Phytoremediation 13, 592–600.
545
Sriprapat, W., Koompai, S., Techkarnjanaruk, S., Thiravetyan, P., 2011. Diethyleneglycol re-
moval by Echinodorus cordifolius (L.): the role of plant-microbeinteractions. J. Hazard.
Mater. 185, 1066–1072.
Srivastava, P., Yadav, A.K., Mishra, B.K., 2015. The effects of microbial fuel cell integration
into constructed wetland on the performance of constructed wetland. Bioresour.
Technol. 195, 223–230.
Srivastava, P., Dwivedi, S., Kumar, N., Abbassi, R., Garaniya, V., Yadav, A.K., 2017. Performance
assessment of aeration and radial oxygen loss assisted cathode based integrated con-
structed wetland-microbial fuel cell systems. Bioresour. Technol. 244, 1178–1182.
Strik, D.P., Snel, J.F., Buisman, C.J., 2008. Green electricity production with living plants and
bacteria in a fuel cell. Int. J. Energy Res. 32, 870–876.
Teamkao, P., Thiravetyan, P., 2015. Phytoremediation of mono-, di-, and triethyleneglycol
by Echinodorus cordifolius L. Griseb. Int. J. Phytoremediation 17, 93–100.
Torit, J., Siangdung, W., Thiravetyan, P., 2012. Phosphorus removal from domestic waste-
water by Echinodorus cordifolius L. J. Environ. Sci. Health A 47, 1–13.
Türker, O.C., Yakar, A., 2017. A hybrid constructed wetland combined with microbial fuel
cell for boron (B) removal and bioelectric production. Ecol. Eng. 102, 411–421.
Wang, H., Ren, Z.J., 2013. A comprehensive review of microbial electrochemical systems
as a platform technology. Biotechnol. Adv. 31, 1796–1807.
Wang, J., Song, X., Wang, Y., Bai, J., Bai, H., Yan, D., Cao, Y., Li, Y., Yu, Z., Dong, G., 2017. Bio-
electricity generation, contaminant removal and bacterial community distribution as
affected by substrate material size and aquatic macrophyte in constructed wetland-
microbial fuel cell. Bioresour. Technol. 245, 372–378.
Wang, X., Tian, Y., Liu, H., Zhao, X., Peng, S., 2018. Optimizing the performance of organics
and nutrient removal in constructed wetland–microbial fuel cell systems. Sci. Total
Environ. 653, 860–871.
Wang, X., Tian, Y., Liu, H., Zhao, X., Peng, S., 2019. The influence of incorporating microbial
fuel cells on greenhouse gas emissions from constructed wetlands. Sci. Total Environ.
656, 270–279.
Wu, S., Lv, T., Lu, Q., Ajmal, Z., Dong, R., 2016. Treatment of anaerobic digestate superna-
tant in microbial fuel cell coupled constructed wetlands: evaluation of nitrogen re-
moval, electricity generation, and bacterial community response. Sci. Total Environ.
580, 339–346.
Xu, L., Zhao, Y., Doherty, L., Hu, Y., Hao, X., 2016. The integrated processes for wastewater
treatment based on the principle of microbial fuel cells: a review. Crit. Rev. Environ.
Sci. Technol. 46, 60–91.
Xu, L., Zhao, Y., Wang, X., Yu, W., 2018a. Applying multiple bio-cathodes in constructed
wetland-microbial fuel cell for promoting energy production and bioelectrical de-
rived nitrification denitrification process. Chem. Eng. J. 334, 105–113.
Xu, F., Cao, F., Kong, Q., Zhou, L., Yuan, Q., Zhu, Y., Wang, Q., Du, Y., Wang, Z., 2018b. Elec-
tricity production and evolution of microbial community in the constructed wetland-
microbial fuel cell. Chem. Eng. J. 339, 479–486.
Xu, G., Zheng, X., Lu, Y., Liu, G., Luo, H., Li, X., Zhang, R., Jin, S., 2019. Development of mi-
crobial community within the cathodic biofilm of single-chamber air-cathode micro-
bial fuel cell. Sci. Total Environ. 665, 641–648.
Yadav, A.K., Dash, P., Mohanty, A., Abbassi, R., Mishra, B.K., 2012. Performance assessment
of innovative constructed wetland-microbial fuel cell for electricity production and
dye removal. Ecol. Eng. 47, 126–131.