Abstract
Bio-photovoltaic devices (BPVs) harness photosynthetic organisms to
produce bioelectricity in an eco-friendly way. However, their low energy
efficiency is still a challenge. A comprehension of metabolic
constraints can result in finding strategies for efficiency enhancement.
This study presents a systemic approach based on metabolic modeling to
design a regulatory defined medium, reducing the intracellular
constraints in bioelectricity generation of Synechocystis sp.
PCC6803 through the cellular metabolism alteration. The approach
identified key reactions that played a critical role in improving
electricity generation in Synechocystis sp. PCC6803 by comparing
multiple optimal solutions of minimal and maximal NADH generation using
two criteria. Regulatory compounds, which controlled the enzyme activity
of the key reactions, were obtained from the BRENDA database. The
selected compounds were subsequently added to the culture media, and
their effect on bioelectricity generation was experimentally assessed.
The power density curves for different culture media showed the BPV fed
by Synechocystis sp. PCC6803 suspension in BG-11 supplemented
with NH4Cl achieved the maximum power density of 148.27
mW m-2. This produced power density was more than
40.5-fold of what was obtained for the BPV fed with cyanobacterial
suspension in BG-11. The effect of the activators on BPV performance was
also evaluated by comparing their overpotential, maximum produced power
density, and biofilm morphology under different conditions. These
findings demonstrated the crucial role of cellular metabolism in
improving bioelectricity generation in BPVs.
Keywords: Regulatory defined medium; Polarization;
Cyanobacteria; Genome-scale metabolic modeling; Photosynthetic
Introduction
Discovering the inherent capability of microorganisms to generate
bioelectricity has opened a new era of renewable energy production.
Regarding the emergence of microbial fuel cells (MFCs) as a system to
extract electrons from the complex organic substrate during the
anaerobic biodegradation process, the critical role of particular
microorganisms as biocatalysts in wastewater treatment and energy
generation has become crystal clear (Slate, Whitehead, Brownson, &
Banks, 2019). Although the MFCs are highly in academic interests
(Santoro, Arbizzani, Erable, & Ieropoulos, 2017), the advantages of
bio-photovoltaic devices (BPVs), which can operate without organic
sources and carbon dioxide emission, reveal more benefits in comparison
with MFCs encountering difficulties of supplying organic feed and carbon
dioxide management (Gul & Ahmad, 2019). In BPVs, the oxygenic
photosynthetic organism is being used as a bio-anode catalyst, deriving
electrons from water photolysis under the light emission (Qi, Ren,
Liang, & Wang, 2018).
Despite the high potential of BPVs as a promising power generator, the
main bottleneck restricting the commercial way is the intense
competition of intracellular pathways for energy resources and intrinsic
metabolic losses resulting in low power outputs generation (McCormick et
al., 2011). Therefore, numerous researches were undertaken to understand
the intracellular electron
pathways of photosynthetic organisms. Among unicellular photosynthetic
organisms, Synechocystis sp. PCC 6803 (henceforth referred to asSynechocystis ), the well-known model organism, received a great
deal of attention to investigate the electron transfer pathways (Thiel
et al., 2019). In this regard, investigation of inhibitors’ effects on
the photosynthetic metabolic pathways (Bombelli et al., 2011), creation
of mutant strains to redirect electron flux (Bradley, Bombelli,
Lea-Smith, & Howe, 2013), and eliminating photosystem II (Cereda et
al., 2014), has been demonstrated in previously published studies to
understand electrons generation mechanism in Synechocystis .
Bombelli et al. investigated the use of classic inhibitors such as DCMU,
DBMIB, and methyl viologen (MV), to identify the electron transfer chain
suggesting that photo-electrons are originated from photolysis of water
and end with the reducing form of PS I (Bombelli et al., 2011). Bradley
et al. redirected electron flux from competing electron sinks by
creating mutant strains of Synechocystis lacking respiratory
terminal oxidase complexes and using ferricyanide as an electron
acceptor. When terminal oxidase complexes were inactivated, 10% of the
respiratory electron flux was redirected to ferricyanide in the dark
condition (Bradley et al., 2013). Cereda et al. studied the effect of
photosystem II (PS II) on electron generation through both inhibitor
addition and gene deletion. It was also proved that the primary
photocurrent generation requires the activation of PS II during water
photolysis (Cereda et al., 2014). Moreover, their case study on theSynechocystis revealed the incapability of transferring electrons
to the electrode owing to numerous metabolic pathways facing fierce
competition with the electrode for the electrons. Thus, extracellular
photo-electrons are attributed to a small proportion of the total
organism electron flux (Cereda et al., 2014). This matter has been
pointed out in Glazier’s work that cellular metabolism used only 3% of
electrons produced by photosynthetic light reactions (Glazier, 2009),
and thus the metabolism has considerable potential for electricity
generation. However, switching the cellular metabolism to the generation
of NADH is required.
Despite the careful investigations on the photosynthetic electron
pathways, the importance of a system-oriented strategy applying
metabolic modeling to uncover other metabolic pathways that may
contribute to electrons production has been neglected. Mao and Verwoerd
applied a metabolic network using flux balance analysis (FBA) to explore
the inherent capacity of electron generation for Synechocystis ;
however, the lack of experimental works in their studies is evident (Mao
& Verwoerd, 2013).
Enhancing intracellular electron carriers such as NADH and
NAD+ can be considered a positive approach to
enhancing energy generation in bioelectrochemical systems. In this
regard, Han et al. regenerated NADH by genetically engineeredClostridium ljungdahlii , and increased the maximum power density
of the MFC more than 2-fold (Han, Gao, Ying, & Zhou, 2016). Moreover,
It was demonstrated that the electricity generation ofSynechocystis depended on NADH production, which is profoundly
affected by various metabolic pathways (Mao & Verwoerd, 2013).
Therefore, concerning the vital role of metabolic modeling to identify
intracellular constraints and switch the cellular metabolism to the
generation of NADH, a regulatory defined medium can be developed to
shift the cell metabolism to the object of interest (Motamedian,
Sarmadi, & Derakhshan, 2019).
In this study, a system-oriented strategy based on metabolic modeling is
used to design a regulatory defined medium, reducing the intracellular
constraints in the bioelectricity generation of Synechocystis via the
cellular metabolism alteration to improve the NADH generation. A
comprehensive investigation is achieved to identify the key reactions
that play a critical role in enhancing electricity production inSynechocystis via a linear algorithm for finding multiple optimal
solutions (LAMOS) (Mekanik, Motamedian, Fotovat, & Jafarian, 2019).
Consequently, the predicted reactions are regulated by adding enzyme
regulators to the culture medium. These compounds regulate the enzyme
activity of the target reactions and are found in the BRENDA database
(Jeske, Placzek, Schomburg, Chang, & Schomburg, 2019). Finally,
regulators’ effect on BPV performance is evaluated by comparing
polarization and power density curves under various conditions.
Material and methods
Metabolic model and in silico simulation
A genome-scale metabolic network of Synechocystis , named iJN678
(Nogales, Gudmundsson, Knight, Palsson, & Thiele, 2012), was used to
investigate the metabolic pathways. The COBRA Toolbox and the GLPK
package were applied to MATLAB 2017b to run the metabolic modeling
(Schellenberger et al., 2011). Optimizing the intracellular electrons
generation rates was done by defining the NADH production rates as the
objective function. The lower bounds of intracellular reversible and
irreversible reactions were applied to -1000 and 0 mmol/gDCW/h,
respectively. Moreover, the upper bounds of all intracellular reactions
were set to 1000 mmol/gDCW/h. Photoautotrophic conditions were simulated
by constraining the photon uptake rate to 52 mmol/gDCW/h, and
bicarbonate uptake rates were set to 3.7 mmol/gDCW/h as the only carbon
source. The lower and upper bounds of exchange reactions including
Co2+, Fe2+, Fe3+,
H+, Na+, Ni2+,
Cu2+, Zn2+, Ca2+,
Mg2+, Mn2+, O2,
H2O, Molybdate, Potassium, Nitrate, Phosphate, and
Sulfate remained unconstrained by -1000 and 1000 mmol/gDCW/h,
respectively. Hence the uptake and secretion of the mentioned
metabolites were allowed (Nogales et al., 2012).
Identifying effective reactions and designing the regulatory
defined medium
As previously described (Mekanik et al., 2019), LAMOS was employed to
calculate multiple optimal flux distributions and determine effective
reactions (Motamedian & Naeimpoor, 2018). For this, the optimal biomass
formation rate was calculated to be 0.0884 mmol/gDCW/h, and the growth
rate was initially bound to 90% of the optimal growth rate; moreover, a
threshold of 10-8 was considered to determine active
reactions. Thus, 10,000 optimal solutions were found for two conditions,
including the maximum and the minimum rates of NADH production (i.e.,\(\text{NADH}\rightarrow\text{NAD}^{+}+H^{+}+2e^{-}\)). Then, for
each mentioned condition, the flux variability for each reaction was
determined (Fig. 1), and two criteria were applied to identify the
candidate reactions for up or down regulations.
As demonstrated in Fig.1, a reaction is suitable to be up-regulated if
its minimum flux in the maximum case (i.e., maximum NADH production
rate) is higher than the maximum flux in the minimum case (i.e., minimum
NADH production rate). In contrast, a reaction is proper to be
down-regulated if its minimum flux in the minimum case is higher than
the maximum flux in the maximum one.
The activity difference for each reaction was regarded as the second
criterion and was defined as a fraction of entire optimal solutions
wherein the reaction was indicated to be active. Therefore, those
reactions were taken into consideration that possessed the absolute
value of activity difference equal to 1. In this regard, the activity
difference of 1 for a reaction indicated that the reaction was entirely
active in every 10,000 optimal solutions of maximum NADH production
while it was completely inactive in all optimal solutions of minimum
NADH production. On the other hand, the reaction, which activated and
inactivated entirely in every 10,000 optimal solutions of minimum and
maximum NADH production, respectively, possessed the activity difference
of -1.
(Fig. 1)
The effect of up (down)-regulation of the predicted reactions was
experimentally investigated by adding regulators to the culture medium.
These regulatory compounds found in the BRENDA database affected the
target enzyme activity controlling the target reaction, which is
predicted to result in the overproduction of NADH and, consequently,
electricity enhancement. Therefore, the investigation of the regulators’
impact on electrons overproduction by Synechocystis is another
goal of the study.
Bio-photovoltaic cell (BPV) assembly
The air-cathode single chamber BPV with a working volume of 8.8
cm3, height of 2.2 cm, and electrodes surface of 4
cm2 was fabricated. The anodic and cathodic chambers
were fabricated from polymethyl methacrylate (PMMA). The graphite-coated
stainless steel mesh (mesh size 400) was used as the anode and cathode
electrodes (Naraghi, Yaghmaei, Mardanpour, & Hasany, 2015). The cathode
was treated by the procedures described in (Cheng, Liu, & Logan, 2006)
to attain 0.5 mg cm-2 Pt loading. A schematic of the
BPV is shown in Fig. 2.
(Fig. 2)
Microorganism and culture media
Synechocystis sp. PCC6803 (subsequently referred to asSynechocystis ) was obtained from Ariyan Gostar Research
Corporation. All cultivation experiments conducted in the BG-11 medium
described in the work of Bombelli et al. (Bombelli et al., 2011). In
this regard, liquid seed culture was obtained periodically, which was
then used for the inoculation of fresh liquid culture. Subsequently, 2
ml of seed culture was inoculated into 100 ml flasks containing 18 ml of
fresh BG-11. All incubations were carried out at 150 rpm and 29 °C,
which were continuously kept under the illumination of a red light bulb
(630 nm) with 6000 lux lighting. It was demonstrated that red light
wavelengths have a significant impact on maintaining photosynthetic
reactions (Sarcina, Bouzovitis, & Mullineaux, 2006).
Inoculation and BPV operation
In all experiments, after five days of cultivation, cyanobacterial cells
were harvested during exponential growth
(\(\text{OD}_{750\ }=3.5\pm 0.1\)) and centrifuged for 15 min at 3000
g. Then, regarding the experiment to be studied, Synechocystiscells resuspended in fresh BG-11 or regulatory BG-11. With regard to the
regulatory BG-11 medium, the selected regulatory compounds were
supplemented to fresh BG-11 according to the concentrations obtained
from Brenda to evaluate their effect on electricity generation. Thus,
the BPV was inoculated by different culture media types under the batch
mode operation and open circuit conditions. The inoculation continued
until a stable cell potential peak was obtained, indicating successful
microbial enrichment fulfillment and cyanobacterial cells stabilization.
The bioelectrochemical experiments were conducted under 6000 lux
lighting of red light (630 nm). High-intensity lighting and red light
illumination were reported to have a positive effect on increasing power
generation of the bioelectrochemical systems (Madiraju, Lyew, Kok, &
Raghavan, 2012).
Results and discussion
Identifying the effective reactions in NADH production
The appropriate reactions contributing to an increase in the
intracellular NADH generation of Synechocystis were predicted and
shown in Fig. 3A. The reactions depicted on the top possessed the
activity difference of 1. Furthermore, their minimum flux under maximum
NADH production rate is more than their maximum flux under the minimum
NADH production rate (Fig. 1A), and thus there were proposed for
up-regulation. In contrast, down-regulation should be considered for
those reactions revealed on the bottom with the activity difference of
-1 in which their minimum flux under minimum NADH production rate is
more than the maximum flux under maximum NADH production rate (Fig. 1B).
The maximum and minimum NADH production rates were calculated to be 2.1
and 0 mmol/gDCW/h, respectively. The predicted reactions were prepared
in more detail in the Supplementary file (Tables S1 and S2). Moreover, a
complete name of all reactions and metabolites were presented in the
Supplementary file (Tables S3 and S4), respectively.
(Fig. 3)
The predicted effective metabolic pathways for enhancing NADH are
schematically represented in Fig. 3B. Herein, two crucial metabolic
pathways for converting acetate (ac) to acetyl-CoA (accoa) that
possessed a significant activity difference were selected. As shown in
Fig. 3C, acetate could be consumed via the acetyl-CoA synthetase (ACS)
by converting ATP to AMP. Besides, this metabolite can also be used
through a series of reactions, including acetate kinase (ACKr_f) and
phosphotransacetylase (PTAr_b), by conversion of ATP to ADP (Fig. 3C).
Thus, acetate kinase and phosphotransacetylase were recommended to
produce acetyl-CoA, which then enters the TCA cycle and causes NADH to
form via malate dehydrogenase (MDH_f).
The systemic approach also recommended that glycerol 3-phosphate
(glyc3p) played a vital role in NADH generation. As demonstrated in Fig.
3B, a red dashed line that starts with glyoxalate carboligase pathway
(GLXCL) and ends with glycerol kinase (GLYK), consumed three moles NADH
along with one mole of ATP to produce glycerol 3-phosphate. These
pathways occur as a series of reactions that each reaction possesses the
same flux through the metabolic network leading to 0 mmol/gDCW/h NADH
generation (Table 1). Therefore, they are regarded as proper candidates
to be down-regulated. Moreover, glycerol 3-phosphate could also be
produced by glycerol-3-phosphate dehydrogenase (G3PD2_b), which
consumes only one mole of NADH without ATP consumption. Green arrows
show this pathway in Fig. 3B. Glycerol-3-phosphate dehydrogenase
possesses the same reaction rates as the mentioned series reactions but
leads to 2.1 mmol/gDCW/h NADH generation (Table 1). Thus, its
up-regulation should be taken to prevent NADH and ATP loss via other
metabolic pathways.
(Table 1)
A further investigation into metabolic pathways depicted in (Fig. 3B),
showed that the generated glycerol 3-phosphate produces acyl carrier
protein (acp), which is then used to form CoA, which can be utilized by
(PTAr_b). Furthermore, the hypothetical glycerol-3-phosphate
acyltransferase reactions (G3PAT Rxns) comprise nine reactions
generating acyl carrier protein, in which their details were mentioned
in the Supplementary file (Table S5).
Microbial enrichment and open circuit potential (OCP)
monitoring
With regard to the introduced reactions by metabolic modeling in Fig.
3A, phosphotransacetylase and glutamate dehydrogenase were taken into
consideration because of the availability of their regulators. As
reported in Brenda and previously published studies, 40mM
NH4Cl resulted in 3-fold stimulation of
phosphotransacetylase (Brinsmade & Escalante-Semerena, 2007), and 50mM
KCl led to an increase in 170% activity of glutamate dehydrogenase
(Bhuiya et al., 2000). Hence, three suspensions for microbial enrichment
were used including Synechocystis + BG-11, Synechocystis +
BG-11 + 50mM KCl, and Synechocystis + BG-11 + 40mM
NH4Cl. Having concluded an effective biofilm formation
during open circuit conditions (Zhang, Zhu, Li, Liao, & Ye, 2011),
which established a uniform biofilm facilitating substrate diffusion and
electron transfer, the microbial enrichment of the BPV has been done by
monitoring the open circuit potential (OCP). Thus, the OCP evolution of
the three culture media was shown in Fig. 4.
(Fig. 4)
Considering the OCP of the sole culture medium of BG-11, the addition of
activators (i.e., KCl or NH4Cl) bring about a
significant increase in the OCP of the BPV. Since Nernst’s equation
depicting the effective parameters on the electrochemical OCP, the
higher cell potentials strongly depend on the activation of redox
species implying the critical role of KCl and NH4Cl in
this increment.
By observing a noticeable decrease in OCP, the fresh medium was injected
into the BPV, as shown by arrows in Fig. 4. The replacement of the fresh
culture medium with the old one causes an increase in the OCP trend.
Since the BPV operated in the batch mode with high cell density
(\(\text{OD}_{750\ }=3.5\pm 0.1\)), the potential reduction can be
attributed to the depletion of nutrients; thus, fresh medium injection
compensates for the potential drop. This phenomenon was also shown
previously in the work of Madiraju et al. (Madiraju et al., 2012), in
which replenishing minerals led to an increase in power density.
Moreover, for the culture media with activator, this abrupt growth in
the OCP was higher than the sole culture medium of BG-11. A comparison
between Figs. 4A and 4B reveal that the culture medium ofSynechocystis + BG-11 + 40mM NH4Cl obtained the
higher OCP and more prolonged stationary phase. This issue puts the
emphasis on the more impact of this culture medium on the cell potential
compared to the BG-11 + 50mM KCl.
Polarization and power density curves
The effect of external resistance
on the BPV performance fed with various culture media types was
investigated by monitoring current evolution. Fig. 5A depicted the
current evolution of Synechocystis for BG-11, BG-11+KCl, and
BG-11+NH4Cl culture media at 500, 500, and 750 kΩ
external resistances, respectively. The presence of KCl increased the
maximum produced current of the BPV by more than 8.8% compared with its
value when the initial culture medium of BG-11 was fed. The addition of
NH4Cl to BG-11 had a remarkable influence on the current
production and increased the maximum current density of the cell more
than 2-fold compared with the BPV fed by BG-11. This incremental
phenomenon was obtained at a higher external resistance of 750 kΩ, which
was comparable to the other media implying more increase have to be
obtained in the same external resistance (i.e., 500 kΩ).
Additionally, the stationary phase in the current evolution (shown by
two dashed horizontal arrows in Fig. 5A) of the
BG-11+NH4Cl fed BPV was longer compared to the BPV
cultured BG-11 and BG-11+KCl, indicating a stable current density by
extension feed replacement.
(Fig. 5)
The BPV overpotentials (including activation, ohmic, and concentration)
can be investigated by applying different external resistances to
polarize the cell. The polarization curves of four types of BPV feed was
illustrated in Fig. 5B. The abrupt reductions of cell potentials in the
polarization curves of BG-11+Synechocystis and
BG-11+KCl+Synechocystis indicate the high activation
overpotential to extract the electron from the cellular metabolism. This
type of overpotential was remarkably higher than two other
overpotentials (i.e., ohmic and concentration overpotentials)
characterized at the middle and end of polarization curves. Activation
overpotential represents the energy that microbes required to transfer
electrons from their surface to the electrode (Logan et al., 2006).
These high drops corroborate the hypothesis presented in previous
researches (Cereda et al., 2014; McCormick et al., 2011), indicating the
incapability of Synechocystis to transfer electrons from its
membrane due to the competition of numerous metabolic pathways for
energy resources. Moreover, the absence and presence of cyanobacteria in
the BG-11 culture medium demonstrated the incapability ofSynechocystis in generating electrons. Although the addition of
KCl increased the current density, the initial sharp reduction could not
be compensated. However, for the BPV fed
BG-11+NH4Cl+Synechocystis , the cell potential
reduction was exceedingly made up, revealing the impressive role of
NH4Cl to compensate for the activation loss of metabolic
reactions and extract electrons from light and water.
Furthermore, the higher current densities obtained at lower external
resistances show the augmented electron production and accentuate the
stimulation of cell electrogenesis metabolic pathways. Low activation
losses owned to the fact that phosphotransacetylase had a crucial role
in converting acetate to acetyl-CoA, assisting the cell to maintain a
balance between biosynthesis and energy production (El-Mansi, Cozzone,
Shiloach, & Eikmanns, 2006). Therefore, the overexpression of this
enzyme by NH4Cl could facilitate electrons transfer.
Moreover, the occurrence of the overshoot phenomenon resulting from
sudden electron depletion at low external resistances that cannot be
compensated by microorganisms (Hong, Call, Werner, & Logan, 2011) was
observed in the BPV fed with all four types of culture media (Fig. 5B).
The overshoot in the BPV fed with BG-11+KCl+Synechocystis led to
a more than 31 % decrease in the current density (from 0.1913 to 0.1306
mA m-2). The culture medium used NH4Cl
as an activator showed a better performance in compensating electron
depletions and only brought about a 6.6 % decrease in the produced
current. This is another evidence emphasizing the crucial role of
NH4Cl, which improves Synechocystiselectrogenesis metabolic pathways.
The power density curves for three culture media were shown in Fig. 5C
to Fig. 5E. The BPV fed by
BG-11+NH4Cl+Synechocystis achieved the maximum
power density of 148.27 mW m-2, which is more than
40.5-fold of what was obtained for the BPV fed with
BG-11+Synechocystis (Fig. 5C).
The maximum produced power density for BG-11+KCl+Synechocystiswas 10.97 mW m-2 (Fig. 5D). Despite the higher salt
concentration, this power density was 13.5 times lower than the BPV fed
by BG-11+NH4Cl+Synechocystis . Achieving a higher
power density by NH4Cl compared to KCl can be related to
the fact that KCl had an adverse effect on some critical metabolic
pathways, which is discussed in the Supplementary file (Table S6).
Therefore, with regard to the role of KCl in the stimulation of
glutamate dehydrogenase, it cannot be helpful in electrogenesis
metabolism as much as NH4Cl.
Furthermore, to scrutinize the effect of NH4Cl on
diminishing intracellular constraints, Synechocystis was removed
from the BPV. In this case, the maximum produced power density for
BG-11+NH4Cl was 13.72 mW m-2 (Fig.
5E). This power density was more than 10-fold lower than the maximum
power density of the BPV fed with
BG-11+NH4Cl+Synechocystis . The abiotically
produced power density is mainly attributed to the medium salinity
(Logan, 2009; Logan et al., 2006). Therefore, the presence of
NH4Cl results in the metabolic stimulation ofSynechocystis rather than the anolyte conductivity improvement.
Thus, the addition of NH4Cl can play a critical role in
shifting the Synechocystis metabolism to produce more
electricity.
Biofilm Morphology
FESEM micrographs of Synechocystis in BG-11 and
BG-11+NH4Cl were illustrated in Fig. 6. In both cases, a
small portion of cells attached to the anode’s surface. This low biofilm
formation reported here is similar to those investigated by (McCormick
et al., 2011) in which 8.5% of Synechocystis attached to the
anode’s surface made of indium tin oxide-coated polyethylene
terephthalate (ITO-PET). Consequently, the addition of
NH4Cl to BG-11 had a small impact on the biofilm
morphology, and the observed power density enhancement is mainly
attributed to the improvement of electrogenesis metabolic pathways.
(Fig. 6)
Conclusion
The system-oriented strategy based on metabolic modeling discloses the
effective reactions and enzyme activators to enhance the bioelectricity
production in BPVs. These activators could provoke theSynechocystis as a strain engaged in noticeable intrinsic
metabolic losses to reduce its activation overpotential significantly
and accelerate bioelectricity generation.
The overexpression of the phosphotransacetylase by NH4Cl
exceedingly facilitates electrons transfer and stimulate cell
electrogenesis metabolic pathways, which brings about lower activation
losses. Although KCl led to an increase in glutamate dehydrogenase
activity, the experimental results showed that it could not be helpful
in electrogenesis metabolism as much as NH4Cl.
Additionally, the investigation of overshoot occurrence in the BPV
demonstrated the culture medium used NH4Cl as an
activator produced a better performance in compensating electron
depletions. Furthermore, the addition of NH4Cl had a
small effect on the biofilm morphology.
Consequently, unlike the addition of classic inhibitors such as DCMU and
DBMIB, which were used to investigate the source of electrons in the
photosynthetic organism, the supplementation of regulators identified by
the systemic approach had a significant impact on improving electrical
power generation. It is worth to mention that the produced power density
of Synechocystis is still relatively lower than exoelectrogenic
bacteria such as Shewanella or Geobacter . However, this
method can be implemented in enhancing exoelectrogenic bacteria
bioelectricity generation.