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Biotechnology Advances 64 (2023) 108101
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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Biophotovoltaics: Recent advances and perspectives
Huawei Zhu a, b, *, Haowei Wang a, b, Yanping Zhang a, Yin Li a, *
a
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Biophotovoltaics
Extracellular electron transfer
Electron extraction strategy
Synthetic microbial consortia
Biophotovoltaics (BPV) is a clean power generation technology that uses self-renewing photosynthetic micro­
organisms to capture solar energy and generate electrical current. Although the internal quantum efficiency of
charge separation in photosynthetic microorganisms is very high, the inefficient electron transfer from photo­
systems to the extracellular electrodes hampered the electrical outputs of BPV systems. This review summarizes
the approaches that have been taken to increase the electrical outputs of BPV systems in recent years. These
mainly include redirecting intracellular electron transfer, broadening available photosynthetic microorganisms,
reinforcing interfacial electron transfer and design high-performance devices with different configurations.
Furthermore, three strategies developed to extract photosynthetic electrons were discussed. Among them, the
strategy of using synthetic microbial consortia could circumvent the weak exoelectrogenic activity of photo­
synthetic microorganisms and the cytotoxicity of exogenous electron mediators, thus show great potential in
enhancing the power output and prolonging the lifetime of BPV systems. Lastly, we prospected how to facilitate
electron extraction and further improve the performance of BPV systems.
1. Introduction
Solar energy is an infinite energy reservoir, which radiates the
earth’s surface at an annual rate of 120,000 TW (Blankenship et al.,
2011; Lewis and Nocera, 2006). Theoretically, the global annual energy
consumption of approximately 18 TW in 2020 can be met by one and a
half hours of sunlight radiation if the light can be fully utilized (Hole­
chek et al., 2022). Therefore, developing efficient and low-cost solar
energy utilization technology is an important approach for supporting
the sustainable development of human society.
The prevailing technology for solar energy utilization is photovol­
taics (PV), which directly convert solar energy into electricity through
photovoltaic effect of semiconductor materials. Since the first PV solar
cell developed using silicon in 1954 (Chapin et al., 1954), PV has un­
dergone a remarkable improvement in photovoltaic materials and effi­
ciencies during recent decades (Polman et al., 2016). The cumulative
global deployment of PV modules has exceeded 500 GW, which accounts
for nearly 3% of global electricity (Heath et al., 2020). However, largescale deployment of PV modules also raises concerns on environment
compatibility and resource availability, such as the toxic elements
contained in some photovoltaic materials, the poisonous chemicals
involved in manufacturing of PV modules, and the demand for rare
metals (Kavlak et al., 2015; Polman et al., 2016).
Biophotovoltaics (BPV), also known as photomicrobial fuel cells or
microbial solar cells, is an emerging technology of converting solar en­
ergy into electrical energy using photosynthetic microorganisms (Howe
Abbreviations: BPV, biophotovoltaics; PV, photovoltaics; PETC, photosynthetic electron transport chains; PSII, photosystem II; PQ/PQH2, plastoquinones; cyt b6f,
cytochrome b6f complex; PC, plastocyanin; PSI, photosystem I; Fd, ferredoxin; FNR, ferredoxin-NADP+ reductase; EET, extracellular electron transfer; MV, methyl
viologen; HNQ, 2-hydroxy-1,4-naphthoquinone; COX, cytochrome c oxidase; Cyd, bd-quinol oxidase; ARTO, alternative respiratory terminal oxidase; FDPs, fla­
vodiiron proteins; OmcS, outer membrane cytochrome S; IET, interfacial electron transfer; FTO, fluorine doped tin oxide; TiO2, titanium dioxide; IO-ITO, inverse
opal‑indium tin oxide; BP-ITO, branched micropillars of ITO nanoparticles; CNTs, carbon nanotubes; rGO, reduced graphene oxide; Os, osmium; PEDOT, poly(3,4ethylenedioxythiophene); SHE, standard hydrogen electrode; BQ, 1,4-benzoquinone; DMBQ, 2,6-dimethyl-1,4-benzoquinone; 2,6-DCBQ, 2,6-dichlorobenzoquinone;
2,5-DCBQ, 2,5-dichlorobenzoquinone; PPBQ, p-phenylbenzoquinone; PYO, pyocyanin; STM, scanning tunneling microscopy; AFM, atomic force microscopy; ECM,
external culture medium; CRISPRi, CRISPR interference.
* Corresponding authors at: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of
Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
E-mail addresses: [email protected] (H. Zhu), [email protected] (Y. Li).
https://doi.org/10.1016/j.biotechadv.2023.108101
Received 13 October 2022; Received in revised form 2 January 2023; Accepted 15 January 2023
Available online 18 January 2023
0734-9750/© 2023 Elsevier Inc. All rights reserved.
H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
and Bombelli, 2020; Wey et al., 2019). Compared with PV technology,
BPV is more environmentally friendly due to the photosynthetic mate­
rials are non-toxic and renewable. In addition to using light to generate
electricity during daytime, BPV systems are hypothesized to generate
electrical current in the dark by oxidizing the intracellular metabolites
(Tanaka et al., 1985), whereas PV systems do not generate power in the
night. Moreover, BPV systems can be designed as an energy-storage
reservoir – a rechargeable battery where the charging and discharging
process can be separated (Liu and Choi, 2019b, 2020; Pankratova et al.,
2022; Ter Heijne et al., 2021), and this feature is more superior to PV
which cannot store electricity. BPV technology thus receives more at­
tentions recently due to these advantages. Nevertheless, the power
output of BPV systems was lag far behind practical applications. The key
challenge of a BPV system is the difficulty of extracting the electrons
from the photosynthetic electron transport chains (PETC) into an
extracellular electrode. Previous review articles systematically discussed
the electrochemical architectures and experimental designs used in BPV
research (Tschortner et al., 2019; Wey et al., 2019). However, a detailed
overview on electron extraction strategies is lacking, although exoge­
nous mediators approach has been systematically discussed previously
(Weliwatte et al., 2021). This article will give an overview on the history
and recent advances of BPV systems, followed by a summary of the
electron extraction strategies, highlighting the recently reported new
findings on the mechanisms of photosynthetic electron extraction.
Lastly, we prospected how to further improve BPV performance through
facilitating electron extraction.
can be directly extracted from PSII/PSI in the form of excited electrons,
or indirectly extracted from reduced energy carriers including PQH2,
NADPH and carbon-fixation products through oxidizing reactions. The
detailed strategies and pathways for electron extraction will discussed
below in Section 5.
Once photosynthetic electrons get away from photosynthetic mi­
croorganisms, the subsequent BPV processes are carried out in a bio­
electrochemical system (Fig. 1), where the electrons are firstly received
by an anode. Meanwhile, a relatively high-potential reaction such as
oxygen reduction takes place at the cathode, and the resulting potential
difference between the anode and cathode drives electrons flow through
an external circuit, thus forming a light-dependent electrical current.
The generated electrical current can be used to run external loads
including pure resistances or electronic facilities, such as a micropro­
cessor used in Internet of Things (IoT) (Bombelli et al., 2022). For
fundamental study and quantitative analysis of a BPV system, threeelectrode systems harboring a reference electrode were also used by
the BPV communities, and the interested readers are suggested to a
recent review on this topic (Wey et al., 2019). The electrical output of a
BPV system is commonly evaluated by current (or current density) or
power (or power density).
3. The history of BPV development
In 1970s, isolated sub-cellular photosynthetic components were used
as the alternative photosensitizers in organic photovoltaics for photo­
current generation. The utilized sub-cellular photosynthetic components
included chlorophyll a (Tang and Albrecht, 1975), PS I (Gross et al.,
1978), thylakoid membranes (Allen and Crane, 1976), chloroplasts
(Haehnel and Hochheimer, 1979) and the bacterial photosynthetic re­
action center (Janzen and Seibert, 1980). For example, Ochiai et al.
(1979) fabricated a photocell by entrapping the isolated chloroplast
with polyvinyl alcohol and deposited it on an SnO2 optically transparent
electrode. This photocell could produce a photocurrent of 1 μA/cm2 on
illumination, and chloroplast PETC was considered to be responsible for
the photocurrent production. These pioneering works demonstrated
photosynthetic materials could convert light energy into electric energy,
but maintaining the activity of these isolated materials under illumi­
nated conditions is a great challenge.
In 1980s, the first BPV system using intact photosynthetic microor­
ganisms was reported (Ochiai et al., 1980). This BPV system was built by
immobilizing the thermophilic algal cells of Mastigocladus laminosus on
the SnO2 electrode. The formed living electrode supported a photocur­
rent production for over 20 days upon continuous illumination, though
the energy efficiency was very low. When another intact thermophilic
cyanobacterium of Phormidium sp. was adopted, an enhanced photo­
current of 6.6 μA/cm2 could be obtained by drying the algal film at
2. The principles of BPV
BPV relies on oxygenic photosynthesis occurring in photosynthetic
microorganisms (Bombelli et al., 2011; Pisciotta et al., 2010). In nature,
photosynthetic organisms include oxygenic phototrophs and anoxygenic
phototrophs. Only oxygenic phototrophs perform oxygenic photosyn­
thesis which uses water as electron donor, thanks to the photosynthetic
apparatus where photosystem II (PSII) and photosystem I (PSI) work
together (Fischer et al., 2016). Oxygenic photosynthesis is an energy
transduction process involving light absorption, photoinduced charge
separation, electron transfer, and electron storage. Firstly, photosyn­
thetic electrons are produced by PSII through water photolysis. The
photosynthetic electrons are then transferred through PETC by passing
plastoquinones (PQ/PQH2), cytochrome b6f complex (cyt b6f), plasto­
cyanin (PC), PSI, ferredoxin (Fd), and eventually converted into NADPH
by ferredoxin-NADP+ reductase (FNR) for carbon fixation and cell
growth. To achieve photoelectric conversion in BPV systems, an addi­
tional step of extracting electrons from intracellular into extracellular
electrode, also referred to exoelectrogenesis or extracellular electron
transfer (EET), is required. Theoretically, the photosynthetic electrons
Fig. 1. A brief schematic representation of BPV sys­
tems. The photosynthetic electrons derived from
photocatalytic water-splitting by photosystem II can
be directly transferred into the extracellular anode, or
indirectly transferred into the anode through two
extra processes of CO2 fixation and organics oxida­
tion. Once received by the anode, the electrons would
flow towards the cathode through an external circuit.
In the cathode, a relatively high-potential reduction
reaction such as oxygen reduction would take place to
consume electrons.
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H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
Yagishita et al., 1998) on photocurrent generation.
Since around 2010, more efforts devoted to exploring BPV technol­
ogy. Firstly, the exogenous mediators-dependent BPV system was stud­
ied quantitatively. Bombelli et al. (2011) established a multi-channel
BPV device with potassium ferricyanide as electron mediator, which
was used for quantitative analysis of key factors that influence the power
output, including cell density, light density, and mediator concentration.
Moreover, a systematic investigation with inhibitor as probes suggested
that the export of electrons originated at the downstream of PSI. Due to
the potential cytotoxicity and photochemical activity of exogenous
mediators, mediator-less BPV systems were developed. By growing
photosynthetic biofilms on a transparent anode, four algal and cyano­
bacterial strains showed the light-dependent exoelectrogenic activity,
moderate temperature (Ochiai et al., 1983). Tanaka et al. (1985)
investigated the source of electrical current. They ascribed the electrical
current produced in the dark to endogenous glycogen, whereas the
electrical current produced in the light mainly came from photosyn­
thetic oxidation of water. In these works, the exogenous electron me­
diators such as methyl viologen (MV) and 2-hydroxy-1,4naphthoquinone (HNQ) were commonly added to the BPV systems for
assisting electron extraction. In summary, the BPV research in the period
of 1980s ~ 2000s mainly focused on the effects of experimental condi­
tions including light (Tanaka et al., 1988; Yagishita et al., 1997), in­
hibitors (Pils et al., 1997; Yagishita et al., 1993), mediators (Martens and
Hall, 1994; Ochiai et al., 1983), electrolytes (Erabi et al., 1995; Ochiai
et al., 1983) and immobilization methods (Yagishita et al., 2000;
A
B
e-
e-
R
R
Light
A
N
O
D
E
e
-
electrons
pools
e-
Light
C
A
T
H
O
D
E
A
N
O
D
E
e-
C
A
T
H
O
D
E
electrons
pools
e-
Molecular level (biotic)
Cellular level (biotic)
I. Redirecting intracellular electron transfer
II. Broadening available photosynthetic microbes
Blocking electron dissipation pathways: Flv2/Flv3, Cyd, COX, ARTO
Cyanobacteria: Synechocystis, Synechococcus, Nostoc, etc
Creating electron export conduits: OmcS
Eukaryotic algae: Chlamydomonas, Chlorella, Dunaliella, etc
D
C
-
e-
R
R
e
Light
A
N
O
D
E
e-
electrons
pools
e-
Light
C
A
T
H
O
D
E
A
N
O
D
E
Cell-electrode interface level (abiotic)
e-
electrons
pools
e-
C
A
T
H
O
D
E
System level (abiotic)
III. Reinforcing interfacial electron transfer
IV. Design of high-performance devices
Design of three-dimentional electrodes with advanced structures
Device miniaturization: higher mass-transfer, lower internal resistance
Surface modification by using nanomaterials and redox polymers
Non-liquid / flat device: flexible in assembly and scale-up of systems
Fig. 2. Graphical representation of different approaches for enhancing electrical outputs of BPV systems. (A) Redirecting intracellular electron transfer by blocking
electron dissipation pathways or creating electron export conduits. (B) Broadening available photosynthetic microorganisms by seeking or screening electroactive
cyanobacteria and algae. (C) Reinforcing interfacial electron transfer by developing advanced electrode architecture or modifying electrode surface using nano­
materials and redox polymers. (D) Design of high-performance devices with different configurations, e.g. device miniaturization.
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H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
with the largest power output of 10.3 mW/m2 recorded from Synecho­
coccus sp. WH 5701 (McCormick et al., 2011). This study further
demonstrated power output was derived from photosynthesis, which
inferred from the consistence between photosynthetic oxygen evolution
rate and power density. Similarly, another ten different genera of cya­
nobacteria all showed light-dependent power output in the absence of
exogenous mediators (Pisciotta et al., 2010), which indicated the lightdependent exoelectrogenic activity might be ubiquitous in cyanobac­
teria. The exoelectrogenic activity may represent a protective mecha­
nism of cyanobacteria under high intensity of light (Pisciotta et al.,
2011).
The maximum power output that can be achieved by BPV technology
is a question remains to be addressed. A theoretical calculation reported
that the achievable power outputs of a BPV system under natural envi­
ronmental conditions could reach up to 7.7 W/m2, which corresponds to
a current density of 24 A/m2 and an energy conversion efficiency of
2.9% (McCormick et al., 2015). Ryu et al. (2010) inserted a nano­
electrode into the algal chloroplast for direct extraction of photosyn­
thetic electrons, resulting in the harvesting of ~20% of the predicted
total photosynthetic electrons with a current density of 20 A/m2. The
calculated photocurrent of a single photosynthetic cell is 1.2 pA, which
is comparable to the exoelectrogenic bacterium of Geobacter with or­
ganics as electron donor (Choi, 2015; Jiang et al., 2013). Although this
nanotechnology is not suitable for scale-up, such a high photocurrent
per single cell demonstrated the great potential of power generation by
BPV technology.
which was a 2-fold increase when compared to that of the wild-type.
Nevertheless, the respective contribution of FDPs and terminal oxi­
dases to EET remains to be further clarified.
Although the EET pathway consisted of multiheme cytochromes does
not exist in cyanobacteria based on genomic evidence (McCormick et al.,
2015), heterogeneous reconstruction of existing EET pathways, such as
the MtrCAB pathway from Shewanella and OMCs pathway from Geo­
bacter (Kracke et al., 2015), might be feasible through synthetic biology
approach. An outer membrane cytochrome S (OmcS) from Geobacter
sulfurreducens was overexpressed in cyanobacterium Synechococcus
elongatus PCC 7942 (hereafter referred to as S. elongatus), which resulted
in a 9-fold improvement of photocurrent (Sekar et al., 2016). The het­
erologous expression of OmcS protein in an engineered nitrogen-fixing
S. elongatus improved ammonia bioelectrosynthesis by approximately
13-fold, and the mechanism was proposed as the transmembrane elec­
tronic communication between electrode and living cells created by
OmcS (Dong et al., 2021). In another study, overexpression of OmcS
protein in Synechococcus elongatus UTEX 2973 increased intracellular
NADH concentrations, thereby enhancing D-lactate production (Meng
et al., 2021). These studies demonstrated the heterologous expression of
multiheme proteins in cyanobacteria could rewire the intracellular or
extracellular electron transfer pathways for different purposes. Although
a single OmcS was introduced successfully, the introduction of a full
functional heterologous EET pathway in photosynthetic microorganisms
has not been reported yet. The potential challenges include posttranslational modification, protein folding, membrane targeting, pro­
tein complex assembly and heavy metabolic burden (Schuergers et al.,
2017).
4. Recent advances in enhancing electrical outputs of BPV
In the last decade, both biotic and abiotic engineering approaches
were used to enhance electrical outputs of BPV systems. These ap­
proaches mainly included: redirecting intracellular electron transfer
(molecular level), broadening available photosynthetic microorganisms
(cellular level), reinforcing interfacial electron transfer (cell-electrode
interface level) and design high-performance devices with different
configurations (system level) (Fig. 2).
4.2. Broadening available photosynthetic microorganisms
The biological materials used in biophotovoltaic studies mainly
include cyanobacteria and eukaryotic algae. The mainstream species
used in recent three years did not change a lot when compared to those
summarized in previous review (Wey et al., 2019). Among cyanobac­
teria strains, the model strain Synechocystis was most frequently used
(Bombelli et al., 2011; Bombelli et al., 2015; Bombelli et al., 2022;
Bradley et al., 2013; Cereda et al., 2014; Chen et al., 2022; Clifford et al.,
2021; Hatano et al., 2022; Kusama et al., 2022; Lai et al., 2021; Madiraju
et al., 2012; McCormick et al., 2011; Reggente et al., 2020; Saar et al.,
2018; Tanaka et al., 2021; Thirumurthy et al., 2020; Wey et al., 2021;
Zou et al., 2009). Another commonly used cyanobacterium is the genera
of Synechococcus (Ciniciato et al., 2016; Gonzalez-Aravena et al., 2018;
Ng et al., 2018; Okedi et al., 2020; Pankan et al., 2020; Torimura et al.,
2001). The representative species is S. elongatus, which is rod-shaped
whereas Synechocystis is coccoid. The morphology of cyanobacteria
may influence the biofilm formation and eventually the power output
(Okedi et al., 2020; Wey et al., 2021). Other species include Spirulina
platensis (Lin et al., 2013; Longtin et al., 2021), Nostoc punctiforme (Sekar
et al., 2014; Wenzel et al., 2018), Microcystis aeruginosa (Lemos et al.,
2021; Ma et al., 2015; Park et al., 2020), Anabaena variabilis (Lee et al.,
2020), Gloeocapsopsis sp. (Gacitua et al., 2020), and Leptolyngbia sp.
(Cevik et al., 2019). In cyanobacteria, the electrons transport outside
from the thylakoid membrane need to across two obstructive layers
including cytoplasmic membrane and cell wall. The latter is mainly
composed of peptidoglycan, outer membrane and S-layer (Wey et al.,
2019), which are usually considered insulating.
Green algae and diatoms are two classes of eukaryotic algae pre­
dominantly used in biophotovoltaic studies. The species of green algae
mainly includes Chlamydomonas reinhardtii (Anderson et al., 2016;
Beauzamy et al., 2020a; Kuruvinashetti and Packirisamy, 2022; Kur­
uvinashetti et al., 2021; Lan et al., 2013; Liu et al., 2022; Sayegh et al.,
2019; Sayegh et al., 2021), Chlorella vulgaris (Herrero-Medina et al.,
2022; Karthikeyan et al., 2019; Karthikeyan et al., 2020; Ng et al., 2020;
Pan and Zhou, 2015; Roxby et al., 2020), Choricystis sp. (Cevik et al.,
2020) and Dunalliela salina (Shlosberg et al., 2021b). For diatoms,
4.1. Redirecting intracellular electron transfer
Enlarging the intracellular electron sinks by blocking the electron
dissipation pathways is a conventional strategy to enhance electron
extraction. Aerobic respiration was generally acknowledged as the pri­
mary electron dissipation pathway in electrogenic bacteria. In the model
cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as
Synechocystis), three terminal oxidases are responsible for respiratory
electron flow, including a cytochrome c oxidase (COX), a bd-quinol
oxidase (Cyd), and an alternative respiratory terminal oxidase (ARTO).
The coding genes of these three terminal oxidases were deleted with
different combinations to evaluate their impacts on power generation.
The ferricyanide reduction rate in COX and Cyd double-deletion mutant
increased by 18-fold, when compared with that of the wild-type (Bradley
et al., 2013). A larger improvement of 24-fold was obtained by further
deleting the ARTO. The potential of these mutants was also reflected in
the increased power output of BPV systems, where the peak power
density generated by triple-deletion mutant was 4-fold higher than that
of the wild-type.
Flavodiiron proteins (FDPs) catalyze light-dependent reduction of O2
to H2O in oxygenic photosynthetic microorganisms, which was used for
protecting photosynthetic apparatus from photodamage (Nikkanen
et al., 2021). Consequently, FDPs serve as another electron dissipation
pathway by catalyzing oxygen reduction along with electron consump­
tion. In Synechocystis, FDP isoforms mainly function in the form of
hetero-oligomers of Flv1/3 or Flv2/4 (Nikkanen et al., 2020). Similarly,
an advanced mutant of Synechocystis was constructed by simultaneously
deleting Flv2, Flv3 and three terminal oxidases (Saar et al., 2018). The
maximum power density generated by this mutant reached 540 mW/m2,
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H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
Phaeodactylum tricornutum and Thalassiosira pseudonana were used
(Laohavisit et al., 2015). Due to the presence of chloroplast in eukaryotic
algae, the electrons transport from thylakoid membrane into the extra­
cellular anode need to across three obstructive layers including chlo­
roplast membrane, cytoplasmic membrane and cell wall, which is even
more challenging.
Besides unicellular microorganisms of cyanobacteria and eukaryotic
algae, the marine macroalgae like Ulva was also utilized recently. The
macroalgae-based BPV system produced a light-dependent current
exceeding 50 mA/cm2, which was remarkably greater than the current
densities produced by unicellular photosynthetic microorganisms
(Shlosberg et al., 2022). The authors speculated that this improvement
was a result of the combination of the efficient photosynthesis of Ulva
and the increased conductivity in seawater electrolyte solution. The high
photosynthetic efficiency of Ulva can be attributed to its large surface-tovolume ratio. Moreover, they indeed confirmed that the increased
salinity elevated the photocurrents from Ulva. Compared with pure
culture strains, natural photosynthetic communities may have stronger
environmental tolerances. Using sediment sample and seawater as the
inoculums of anodic chamber, the resulting system generated a current
with circadian rhythm that was consistent with a photosynthetic nature
(Malik et al., 2009). A BPV system with natural pond sediment as
inoculum generated a positive photo-response current, and it did not
require buffers or electron mediators (Zou et al., 2009). Similarly, the
electrochemical activity of two seawater microbial communities
revealed their potential as biocatalysts for conversion of light to elec­
tricity (Darus et al., 2015). To date, approximately more than fifty
species of photosynthetic microorganisms was documented to work in
BPV systems. However, no common conclusion what microbial species is
more suitable or performs best in BPV systems has been achieved.
such as stainless steel, copper, platinum and tin oxide. These materials
met the basic requirements, but were not suitable for the attachment of
photosynthetic microorganisms. The second-generation anodes were
mainly prepared using carbon materials, with different forms such as
carbon felt (Tsujimura et al., 2001), carbon cloth (Cereda et al., 2014),
carbon paper (Sekar et al., 2014), graphite (He et al., 2009), and reduced
graphene oxide (Ng et al., 2014). The carbon-based anodes were wellknown in rough surface, which was beneficial for microorganism
attachment. Beyond that, the advantages also included cheapness,
robustness, and chemical inertness. However, carbon-based anodes were
also limited by opaque trait and low electrical conductivity.
The third-generation anodes were specially designed for photosyn­
thetic microorganisms, being porous and optically transparent. A porous
ceramic anode with millimeter-sized pores was prepared with fluorine
doped tin oxide (FTO) and titanium dioxide (TiO2) (Thorne et al., 2011).
This novel anode enabled formation of the dense algal biofilm, resulting
in a 16-fold higher power output compared to conventional carbonbased anodes. The transparent anodes with millimeter-sized pores
were suitable for eukaryotic algae, whereas the anodes with
micrometer-sized pores could be better matched with the cellular size of
prokaryotic cyanobacteria. The state-of-the-art electrode architecture in
biofilm photoelectrochemistry was the inverse opal‑indium tin oxide
(IO-ITO) with an optimized pore diameter of 10 μm (Fig. 3A) (Zhang
et al., 2018). ITO nanoparticle is a translucent and conductive material,
which was used to make porous electrodes with different pore sizes
(Wenzel et al., 2018). These includes microporous electrode with pore
diameter of 10–40 μm and nanoporous electrode with pore diameter of
10–100 nm. The photocurrent generated by Synechocystis on micropo­
rous or nanoporous anodes was nearly 300 times higher than that on
non-porous anode. The microporous anode allowed cyanobacterial cells
to enter the pore structure and increased the direct contact area between
cells and anode, and nanoporous anode increased the contact points
between cells and anode although it did not allow cells to reside on the
pore structure. Recently, a branched pillars anode (BP-ITO) was created
by aerosol jet printing of ITO nanoparticles (Fig. 3B), which represents
the fourth-generation of high-performance photosynthetic electrode
(Chen et al., 2022). When electrically wired to the cyanobacterium
Synechocystis, this hierarchical pillar array exhibited favorable biocata­
lyst loading, light utilization and electron flux output, ultimately pro­
ducing a mediated photocurrent density of 245 mA/cm2 that close to the
theoretical maximum value. These studies demonstrated that engi­
neering electrode structure is an effective approach to reinforce IET
process.
4.3. Reinforcing interfacial electron transfer
In BPV systems, the anode acts as an electron collector for photo­
synthetic cells. Thus, the interfacial electron transfer (IET) in the contact
surface of photosynthetic cells and anode is one of the crucial steps for
power output. Developing anode materials was one common strategy to
enhance IET process. The basic requirements for anode materials used in
BPV systems are electrical conductivity, electrochemical stability and
biocompatibility. According to the architecture and roughness, the an­
odes used in BPV systems were divided into four generations (Chen
et al., 2022; Wey et al., 2019). The first-generation anodes featured in
smooth surface and were commonly prepared by inorganic materials
A
B
IO-ITO
BP-ITO
Fig. 3. Two state-of-the-art electrodes used in BPV systems. (A) SEM image of inverse opal‑indium tin oxide (IO-ITO) electrode with 10 μm diameter pores. Reprinted
with permission from Journal of the American Chemical Society (Zhang et al., 2018). Copyright 2017 American Chemical Society. (B) SEM image of branched
micropillar ITO (BP-ITO) electrode with a height of 600 μm and diameter of 20 μm. Reprinted with permission from Nature Materials (Chen et al., 2022). Copyright
2022 Springer Nature.
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Biotechnology Advances 64 (2023) 108101
Table 1
Photocurrent improvement by modifying electrode surface using nanomaterial or redox polymer.
Photosynthetic microorganism
Anode
substrate
Nanomaterial & redox
polymer
Photocurrent density (μA/
cm2)
Improvement vs. unmodified
anode
Reference
Nostoc sp. ATCC 27893
Synechococcus sp.
carbon paper
Pt mesh
CNTs
CNTs
3
14 μA
–
7-fold
Chlorella vulgaris
carbon cloth
rGO
–
2-fold
Chlorella vulgaris
gold
Os redox polymer
5
2-fold
Gloeocapsopsis sp. UTEXB3054
Paulschulzia pseudovolvox
Synechocystis sp. PCC 6803
Leptolyngbia sp.
Mixed culture of algae and
cyanobacteria
graphite
graphite
graphite
gold
Os redox polymer
Os redox polymer
PEDOT
P(DtP-Nptyl-NH2)
2.26
0.44
1
0.2
2.4-fold
22-fold
6-fold
8-fold
(Sekar et al., 2014)
(Park and Song, 2021)
(Senthilkumar et al.,
2018)
(Herrero-Medina et al.,
2022)
(Gacitua et al., 2020)
(Hasan et al., 2015)
(Reggente et al., 2020)
(Cevik et al., 2019)
graphite
FcPAMAM
1.18
35-fold
(Cevik et al., 2018)
CNTs: carbon nanotubes; rGO: reduced graphene oxide; PEDOT: poly(3,4-ethylenedioxythiophene); P(DtP-Nptyl-NH2): poly(5-(4H-dithieno [3,2-b:2′ ,3′ -d]pyrol-4-yl)
napthtalane-1-amine); FcPAMAM: ferrocene cored polyamidoamine dendrimer.
density of 0.4 μW/m2 (Chiao et al., 2006). Bombelli et al. (2011) con­
structed a 150 μL miniaturized device that generated a power density of
1.2 mW/m2. Whereafter, a smaller microfluidic device of 0.4 μL was
fabricated using standard soft lithography, allowing power density
increased from 10 mW/m2 to 105 mW/m2 (Bombelli et al., 2015). After
independent parameter optimization for the charging and discharging
processes, a high mediated power density of 500 mW/m2 was generated
in a 40 μL flow-controlled device (Saar et al., 2018). It can be concluded
that the high power densities were more likely to be obtained in mini­
aturized devices.
Choi group worked on the development of miniaturized BPV devices
in recent years. A total of four generations of miniaturized devices were
developed, the size of which ranging from 50 μL to 300 μL. The firstgeneration device was a traditional two-compartment configuration
with a transparent film gold electrode as the anode and a power density
of 0.07 mW/m2 achieved (Yoon et al., 2014). The second-generation
device introduced an air cathode with an air-bubble trap allowing
freely available oxygen, and the anode was designed to maximize light
absorption (Lee and Choi, 2015). The internal resistance of this device
was reduced from 2.3 MΩ in the first generation to 470 KΩ, and the
power density was increased to 9 mW/m2. The improvements of thirdgeneration device reflected in better biofilm formation by using com­
posite graphite anode, smaller internal resistance by designing sandwich
configuration, and quicker gas exchange by inserting a gas permeable
membrane (Wei et al., 2016). The power density was further increased
to 27 mW/m2. Using a three-dimensional conductive polymer-coated
anode, the fourth-generation device with fully gas-permeable configu­
ration was developed, which enabled power density reaching up to 438
mW/m2 (Liu and Choi, 2017a). Moreover, the miniaturized device was
further developed into a self-charging supercapacitor (Liu and Choi,
2019b). The stacking of eight supercapacitors in series allowing output
voltage amplified up to 1.8 V and stability maintained after multiple
charging-discharging cycles.
The BPV systems are generally liquid-based, and semi-solid/solid
systems might be more suitable for scale-up and practical applications.
Sodium alginate hydrogel was used to immobilize algae cells to the
anode, increasing power density by 20% compared with suspension
form (Ng et al., 2017). In addition, an agar-based solid-state BPV system
was fabricated, which allowed versatile device configuration and
permitted easy integration and operation (Mohammadifar et al., 2020).
Hydrogel immobilization makes system stay in a solid state, whereas
paper could serve as base material of anode, allowing system to be flat
and flexible. Sawa et al. (2017) creatively used inkjet printer to fabricate
a ultra-thin biophotovoltaic device on paper, including printing carbon
nanotubes conductive layer on paper and then printing cyanobacteria
layer on the top, achieving rapid integration of biotic and abiotic parts.
Series-parallel arraying of paper-printed BPV devices could power a
small digital clock and a low-power LED light. Another similar paper-
Surface modification for electrode is another approach to strengthen
the electronic communication between cellular surface and electrode.
Several nanomaterials and redox polymers with excellent conductivity
have been used to modify electrode surface for higher photocurrent
generation (Table 1). Carbon nanotubes (CNTs)-modified carbon paper
and reduced graphene oxide (rGO)-modified carbon cloth are appro­
priate for immobilization of photosynthetic microorganisms on the
electrode surface, thus enlarging the interface area and facilitating the
extraction of photosynthetic electrons (Sekar et al., 2014; Senthilkumar
et al., 2018). In addition, the dispersed CNTs could be used as filler of
conductive nanofluid, creating an electron transfer network for sus­
pended cyanobacterial cells (Park et al., 2020; Park and Song, 2021).
Redox polymers are electroactive macromolecules containing localized
sites or groups that can be oxidized and reduced (Casado et al., 2016).
Considering the tunability of redox potential and structure, osmium (Os)
redox polymers have been used for providing electron conduits between
different photosynthetic microorganisms and electrode surfaces (Gaci­
tua et al., 2020; Herrero-Medina et al., 2022). Hasan et al. (2015) used a
series of Os-polymer to optimize the energy gap between the photo­
synthetic cells and polymers. The optimal Os-polymer enabled a 22-fold
photocurrent improvement. Reggente et al. (2020) developed a poly
(3,4-ethylenedioxythiophene) (PEDOT)-modified graphite electrode,
which exhibited a 6-fold improvement on mediator-less photocurrent
over bare graphite. Grattieri et al. (2019) prepared a bio-inspired redox
polymer naphthoquinone-modified linear polyethyleneimine (NQLPEI), which contains redox center mimicking the natural electron
acceptor of PETC. This quinone-based redox polymer allowed EET be­
tween purple bacteria and the electrode surface. To avoid timeconsuming and separate synthesis of redox polymers, Buscemi et al.
(2022) demonstrated that the deposition of purple bacteria with
simultaneous polymerization of dopamine on the electrode surface
enabled a 5-fold enhancement in EET at the biotic/abiotic interface.
Other redox polymers including ferrocene-containing polymer (Cevik
et al., 2018) and dithienopyrrole-containing polymer (Cevik et al.,
2019) also enhanced photocurrent generation by increasing the sites for
electron collection.
4.4. Design of high-performance devices with different configurations
The configuration of an electrochemical device directly affects the
mass transfer, internal resistance, volumetric power density of BPV
systems, and also matters the cost and scale-up. In general, a compact or
miniaturized device can improve mass transfer, reduce internal resis­
tance, and enhance power output. The volume of a typical BPV device
(anodic chamber) is 10–500 mL, while the volume of a miniaturized
device is 0.2–500 μL.
The miniaturization of BPV devices can be dated back to 2006, in
which a microfluidic device with a volume of 16 μL generated a power
6
H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
based BPV system was developed, in which a paper substrate was coated
with a conductive polymer layer and a biocatalyst layer of cyanobac­
teria, thus assembling into a ultra-thin device with maximum current
density and power density of 650 mA/m2 and 107 mW/m2, respectively
(Liu and Choi, 2019a).
thermodynamically unfavorable to capture electrons from PETC (QA:
− 0.14 V; QB: − 0.06 V; PQ/PQH2: +0.08 V; Fd: − 0.41 V; FNR: − 0.38 V;
NADP+/NADPH: − 0.32 V vs. SHE at pH 7.0) (Tschortner et al., 2019).
Quinones are small molecules involved in electron transfer of key
processes of living organisms, including aerobic respiration (ubiqui­
none), anaerobic respiration (methyl naphthoquinone) and photosyn­
thesis (plastoquinone). In view of this, quinones are widely used as
electron mediators in bioelectrochemical systems. 1,4-Benzoquinone
(BQ) and 2,6-dimethyl-1,4-benzoquinone (DMBQ) were found to func­
tion as effective exogenous electron mediators to capture electrons from
photosystems (Torimura et al., 2001). The exogenous quinones might be
able to more preferably capture electrons from QA/QB sites by
competing with PQ due to their structural similarity. The higher po­
tentials of BQ and DMBQ (+0.28 V and +0.32 V vs. SHE, respectively)
than PQ (+0.08 V vs. SHE) makes them more favorable to extract
electrons from photosystems. In addition, 2,6-dichlorobenzoquinone
(2,6-DCBQ), 2,5-dichlorobenzoquinone (2,5-DCBQ) and p-phenyl­
benzoquinone (PPBQ) were found more efficient than BQ and DMBQ to
derive electrons from the photosynthetic chain of green alga
C. reinhardtii (Longatte et al., 2015). The addition of quinones into BPV
systems could considerably increase the photocurrent output, but the
photocurrent appeared to decline over time thereafter, which might be
ascribed to the photoinactivation of photosystem, the kinetic quenching
of quinones and the reduction of PSII open centers (Longatte et al.,
2018). Moreover, the lipophilicity of quinones makes them more likely
to remain inside the cells, thereby limiting the outflow of reductive
mediators (Beauzamy et al., 2020b). Readers interested in learning
quinones-based redox mediation are referred to a recent review (Weli­
watte et al., 2021).
Ferricyanide was also widely used in BPV systems (Anderson et al.,
2016; Bombelli et al., 2011; Bradley et al., 2013; Hasan et al., 2015; Lai
5. Electron extraction strategies in BPV systems
The quantum efficiency of charge separation process occurred in PSII
is close to 100% (Romero et al., 2017), which means the process that
converts solar photons into electrons is efficient. Thus, the EET process
transporting electron outwards becomes the key step of BPV systems. We
categorized the electron extraction strategies into three classes,
including exogenous electron mediators-dependent strategy, intrinsic
exoelectrogenic activity-dependent strategy and synthetic microbial
consortia-dependent strategy (Fig. 4).
5.1. Exogenous electron mediators-dependent strategy
Electron mediators are class of small molecules that can reversibly be
oxidized and reduced (Watanabe et al., 2009). The electron mediators
circularly perform the processes of electron capture (being reduced) and
electron release (being oxidized), thereby facilitating electron transfer
from the microorganisms into an electrode. In early BPV studies, the
introduction of exogenous electron mediators was the main strategy to
achieve electrical wiring between photosynthetic microorganisms and
electrodes. The widely used electron mediators included HNQ (Tanaka
et al., 1988; Yagishita et al., 1993; Yagishita et al., 1998) and MV
(Coetzee and Stevens, 1993; Erabi et al., 1995; Ochiai et al., 1980).
However, the relatively negative redox potentials of HNQ (− 0.14 V vs.
standard hydrogen electrode, SHE) and MV (− 0.45 V vs. SHE) are
A
e-
B
e-
X-
X
e-
e-
X1-
X1
OM
C
e-
Y-
Y
electroactive bacteria
OM
× ×
X1-
X1
Periplasm
e-
energy carriers
(e.g. lactate)
PM
PM
X-
X
e
PSII
TM
e-
Cyt b6f
PSI
e-
Y-
Y
X
e-
e-
-
ATPase
e-
PSII
TM
e-
Cyt b6f
PSI
e-
ebiomass
ATPase
CO2+H2O
2H2O
4H++O2
Exogenous mediators-dependent
2H2O
4H++O2
Intrisic exoelectrogenesis-dependent
Synthetic microbial consortia-dependent
Fig. 4. Three strategies adopted in BPV systems for photosynthetic electrons extraction. (A) Exogenous mediators-dependent strategy. X represents exogenously
added electron mediators, such as 1,4-benzoquinone and pyocyanin. X− represents the reduction state of X. In some cases, the exogenous mediators such as ferri­
cyanide (denoted as X1) take electrons from the periplasmic space of the cell because they cannot permeate the plasma membrane. (B) Intrinsic exoelectrogenic
activity-dependent strategy. As the exact mechanisms remain to be revealed, three putative EET pathways are shown, including nanowires, c-type cytochromes and
endogenous electron mediators. Electrically conductive nanowires and c-type cytochromes-based EET pathways are inserted with a cross symbol as they may not
exist in cyanobacteria according to the latest evidences. Y represents endogenously secreted electron mediators, such as NADPH. Y− represents the reduction state of
Y. (C) Synthetic microbial consortia-dependent strategy. Photosynthetic microorganisms use solar energy to fix carbon dioxide into organic matters, e.g. lactate and
sucrose. These organic matters serve as the energy carriers to be consumed and oxidized anaerobically by electroactive bacteria for electricity generation. The
diagrams showed in (A) and (B) are the cell topology of cyanobacterial cell. Abbreviations: TM: thylakoid membrane; PM: plasma membrane; OM: outer membrane.
PSII: photosystem II; Cyt b6f: cytochrome b6f complex; PSI: photosystem I; ATPase: ATP synthase.
7
H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
et al., 2021; Laohavisit et al., 2015; Thorne et al., 2011; Thorne et al.,
2014). A high power density of 500 mW/m2 was achieved in a BPV
system with ferricyanide as electron mediator (Saar et al., 2018). As a
non-lipid-soluble molecule, ferricyanide is not able to permeate the
plasma membrane. Ferricyanide usually crosses the outer membrane
into the periplasmic space, and then receives electrons at the outside of
the plasma membrane. Due to the membrane impermeability, ferricya­
nide is less cytotoxic than the quinones (McCormick et al., 2015).
Additionally, ferricyanide is highly soluble in water, which allows high
dose addition in BPV systems (Kim et al., 2020). Nevertheless, ferricy­
anide is sensitive to ultraviolet light and would be decomposed to Fe
(OH)3 and KCN (Asperger, 1952), which is highly toxic to microorgan­
isms. Therefore, ferricyanide is not suitable for long-term operation,
especially if operated under full-spectrum light.
Redox potential is an important consideration when choosing elec­
tron mediators. Electron transfer only occurs from a more negative to a
more positive potential based on the thermodynamic feasibility
(Tschortner et al., 2019). In theory, a mediator with higher redox po­
tential has larger thermodynamic driving force to take electrons from
the PETC, but larger potential difference means more energy loss. The
redox potential of a thermodynamically appropriate electron mediator
should be slightly more positive than that of the key component such as
Fd (− 0.41 V vs. SHE) in PETC. Recently, Clifford et al. (2021) used a
phenazine compound of pyocyanin (PYO) as an effective cell-permeable
mediator, resulting in a 4-fold improvement of photocurrent. The redox
potential of PYO (− 0.12 V vs. SHE) is more negative than the commonly
used DCBQ (+0.32 V vs. SHE), which implies a less thermodynamic loss.
Moreover, PYO could be produced endogenously in a genetically engi­
neered cyanobacterium. Thereby, PYO might be a good electron medi­
ator for photosynthetic electron extraction.
The exogenous electron mediators can effectively improve the
photocurrent density, but the use of exogenous mediators would bring
cytotoxicity, environmental pollution and high cost (Hasan et al., 2015;
Schuergers et al., 2017; Xie et al., 2011). Furthermore, exogenous me­
diators ubiquitously show photochemical instability, low diffusion rate
and non-specific targeting (Kim et al., 2020). All these features lead to
the power output of mediator-dependent BPV systems unstable. One
ingenious solution was to confine the redox moieties of mediator in a
polymer backbone, which avoided their unwanted release into the
environment and also showed reduced cytotoxicity with respect to their
monomeric counterpart (Grattieri et al., 2019).
of hexaheme cytochrome OmcS could mediate long-distance electron
transfer (Wang et al., 2019).
The recent findings and understandings in the intrinsic exoelectro­
genic activity of photosynthetic microorganisms were summarized in
Table 2. Analysis of the genome sequence of Synechocystis revealed no ctype cytochromes are present in the cytoplasmic membrane, the peri­
plasmic space or the outside of Synechocystis (McCormick et al., 2015),
indicating a molecular basis different from that of S. oneidensis and
G. sulfurreducens. Gorby et al. (2006) found that cyanobacterium Syn­
echocystis could produce nanowires when cultured under carbon
limiting condition. The electrical conductivity of these nanowires was
proved by scanning tunneling microscopy (STM) and tunneling spec­
troscopy. Furthermore, it was reported that another photosynthetic
microorganism M. aeruginosa PCC 7806 could also produce conductive
nanowires under specific culture conditions (Sure et al., 2015). In
addition, the conductive nanowires produced in Synechocystis was found
belong to type IV pili (Sure et al., 2015). However, a recent study
demonstrated that type IV pili did not play a role in the photocurrent
generation of Synechocystis (Thirumurthy et al., 2020). Simultaneously,
conductivity measurement using atomic force microscopy (AFM) also
revealed that the type IV pili were not conductive. Wey et al. (2021) also
found there was no significant difference of photocurrent (normalized to
the chlorophyll content) between the wild-type and pili mutant of Syn­
echocystis, indicating type IV pili did not contribute to exoelectrogenic
activity. Taken together, the nanowires might not account for the
intrinsic exoelectrogenic activity of cyanobacteria.
Endogenous electron mediators might be responsible for the exoe­
lectrogenic activity of photosynthetic microorganisms. Phenazines, fla­
vins and quinones are three prominent mediators that can be
biologically synthesized and secreted by some microorganisms. How­
ever, these mediators could be excluded because the genes for phenazine
biosynthesis and the secretion systems for transporting flavins and
quinones are not present in cyanobacteria (McCormick et al., 2015).
Zhang et al. (2018) reported that Synechocystis was able to secrete a
compound with a redox potential of +0.34 V vs. SHE when exposed to
light, which might correspond to benzoquinone or flavin derivatives.
Saper et al. (2018) also found that cyanobacteria secreted a watersoluble redox molecule under light, with molecular weight < 3 kD.
The authors hypothesized that this molecule could be a soluble quinone,
a flavonoid or a small peptide. Recently, NADPH was identified as the
major endogenous mediator in cyanobacteria-based BPV systems.
Shlosberg et al. (2021a) found that NADPH can be secreted by Syn­
echocystis and accumulated in the external culture medium (ECM) once
BPV system was running under light condition. Furthermore, the addi­
tion of exogenous FNR to bind NADPH eliminated the photocurrent
generation, while the addition of NADP+ enhanced the photocurrent
generation. Moreover, this NADPH-mediated mechanism was found to
be conserved in other cyanobacteria species, including S. elongatus and
Acaryochloris marina MBIC 11017. Besides cyanobacteria, NADPH was
also involved in the photocurrent generation in microalgae Dunalliela
salina (Shlosberg et al., 2021b) and marine macroalgae Ulva sp.
(Shlosberg et al., 2022). By using several mutants deficient in metabolic
pathway responsible for NADPH production, Hatano et al. (2022)
demonstrated NADPH produced in the dark periods was critical for
photocurrent generation of Synechocystis. However, Kusama et al.
(2022) reported that no NADPH was detected in the ECM regardless of
before and after the electrochemical measurement, and the fluorescence
pattern of ECM has a discrepancy with that of NADPH standard. Taken
together, it remains controversial whether NADPH serves as an endog­
enous electron mediator of cyanobacteria. More in-depth investigations
are required to figure out which endogenous redox molecules are
involved in the exoelectrogenesis of photosynthetic microorganisms.
Cellular structure, especially cell membrane and cell wall, is prob­
ably an important factor that determines the exoelectrogenic activity of
photosynthetic microorganisms. Wey et al. (2021) constructed several
outer layer mutants and sub-cellular fractions of Synechocystis (ΔS-layer
5.2. Intrinsic exoelectrogenic activity-dependent strategy
The photosynthetic microorganisms in BPV systems mainly work in
two forms: suspension and biofilm. The former only functions when
electron mediators are present. Once photosynthetic biofilms form on
the electrode surface, the electron extraction relies on the intrinsic
exoelectrogenic activity. It is conceivable that the stability and the
duration of intrinsic exoelectrogenic activity-dependent BPV systems
can be improved in the absence of cytotoxic exogenous mediators.
Many photosynthetic microorganisms including prokaryotic cyano­
bacteria and eukaryotic algae have been demonstrated to exhibit lightdependent exoelectrogenic activity, but the detailed mechanisms un­
derlying this phenomenon remain undetermined. Shewanella oneidensis
and Geobacter sulfurreducens are two well-studied exoelectrogenic mi­
croorganisms capable of transferring electrons outside cells to solidphase electrode through efficient EET pathways (Logan et al., 2019).
However, both S. oneidensis and G. sulfurreducens are non-photosynthetic
bacteria thus they cannot produce photocurrent. In S. oneidensis, a
protein complex comprising three subunits (MtrA, MtrB, MtrC) formed
an insulated transmembrane molecular wire, and the electron transfer
along this wire was dependent on the heme group in c-type cytochromes
(Edwards et al., 2020). Similarly, G. sulfurreducens also utilizes hemecontaining c-type cytochromes as transmembrane conductive compo­
nents. The nanowires assembled in G. sulfurreducens by polymerization
8
H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
Table 2
Recent findings on the exoelectrogenic mechanisms of photosynthetic
microorganisms.
Mechanism
(1) Nanowires
Cyanobacteria could
produce
electrically
conductive
nanowires
Type IV pili did not
contribute to
exoelectrogenic
activity of
cyanobacteria
Evidence
The conductivity was
confirmed by
scanning tunneling
microscopy and
tunneling
spectroscopy
The conductivity was
confirmed by
conductive atomic
force microscopy
(AFM)
No significant
difference in
photocurrent of
wild-type and pili
mutant (ΔpilD);
Conductivity
measurement by
AFM indicated pili
structure was not
conductive
No significant
difference in
photocurrent of
wild-type and pili
mutants (ΔpilA1 and
ΔpilB1)
Photosynthetic
microorganism
(3) Endogenous electron mediator (NADPH)
NADPH was
identified in ECM by
two-dimensional
fluorescence (2DFM); Exogenously
adding NADP+
increased
NADPH serves as an
photocurrent;
endogenous
Exogenously adding
mediator in
FNR for binding
cyanobacteria
NADPH decreased
photocurrent;
NADPH
accumulation in
ECM was dependent
on light and
electrochemical
measurement
Mechanism
Reference
Synechocystis
(Gorby et al.,
2006)
Synechocystis
M. aeruginosa
(Sure et al.,
2015)
Synechocystis
(Thirumurthy
et al., 2020)
Synechocystis
(Wey et al.,
2021)
(2) Endogenous electron mediator (unidentified)
Cyclic voltammetry
An unidentified
(CV) indicated an
endogenous
redox active species
mediator (waterwas secreted by
Synechocystis
soluble, lightcyanobacteria
induced release,
(iSyn), especially
MW < 3 kD)
under light condition
An unidentified
endogenous
A reversible redox
mediator
wave was observed
Synechocystis
(midpoint
in CV plots
potential of +0.34
V vs. SHE)
Replacement of
supernatant with
An unidentified
fresh BG11
endogenous
electrolyte abolished
Synechocystis
mediator (MW < 3
the high
kD)
photocurrent from
outer membranedeprived cells
Table 2 (continued )
NADPH serves as an
endogenous
mediator in
microalgae
NADPH serves as an
endogenous
mediator in
macroalgae
NADPH-dependent
mechanism was
not applicable
(4) Cellular structure
Surface layer did not
impede the
exoelectrogenic
activity of
cyanobacteria
(Saper et al.,
2018)
(Zhang et al.,
2018)
Periplasmic space
did contribute to
the complexity of
photocurrent
profile
(Kusama
et al., 2022)
Outer membrane
was a barrier
limiting the
exoelectrogenic
activity of
cyanobacteria
Cellular structure
might be a barrier
for mediator
transport
Evidence
Photosynthetic
microorganism
Reference
The mutants
deficient in NADPH
production produced
a lower photocurrent
NADPH was
identified in ECM by
2D-FM; Exogenously
adding NADP+
increased
photocurrent;
Exogenously adding
FNR decreased
photocurrent
NADPH was
identified in ECM by
2D-FM
Synechocystis
(Hatano et al.,
2022)
D. salina
(Shlosberg
et al., 2021b)
Ulva
(Shlosberg
et al., 2022)
NADPH was not
detected in ECM;
There was a
discrepancy of
fluorescence pattern
between the
supernatant and
NADPH; The
fluorescence
intensity of
intracellular NADPH
did not changed
when photocurrent
was increased
Synechocystis
(Kusama
et al., 2022)
No significant
difference in
photocurrent of
wild-type and ΔSlayer mutant
Spheroplast
(periplasmic space
was absent)
exhibited
monophasic
photocurrent profile
Synechocystis
(Wey et al.,
2021)
Synechocystis
(Wey et al.,
2021)
Outer membrane
deprivation led to an
order-of-magnitude
increase of
photocurrent
Synechocystis
(Kusama
et al., 2022)
A gentle physical
treatment for
cyanobacteria
increased
photocurrent
Synechocystis
(Saper et al.,
2018)
Synechocystis: Synechocystis sp. PCC 6803; S. elongatus: Synechococcus elongatus
PCC 7942; M. aeruginosa: Microcystis aeruginosa PCC 7806; A. marina: Acaryo­
chloris marina MBIC 11017; D. salina: Dunalliela salina; Ulva: macroalgae Ulva sp.
Synechocystis
S. elongatus
A. marina
mutant and spheroplasts lacking peptidoglycan layer) and found that the
intact periplasmic space (and outer membrane) resulted in the
complexity of photocurrent profile, but the lacking of periplasmic space
did not significantly enhance the photocurrent. In addition, S-layer did
not impede the photocurrent generation as well. Kusama et al. (2022)
obtained an outer membrane-deprived mutant of Synechocystis by
employing CRISPR interference (CRISPRi) technology. Interestingly, an
order-of-magnitude enhancement in cyanobacterial EET activity was
achieved after detaching the outer membrane. This remarkable
enhancement of EET activity also reflected in the rapid ferricyanide
reduction and electron donation capacity to heterotrophic bacteria.
Another study found that gentle physical treatment of cyanobacteria
(Shlosberg
et al., 2021a)
9
H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
enhanced photocurrent, indicating the intact cellular structure might be
a barrier for the transport of electron mediators (Saper et al., 2018).
These findings verified the hypothesis that the low permeability of the
outer membrane is associated with the low EET activity of cyanobac­
teria. Therefore, weakening cellular structure provides an effective
strategy to enhance electrical outputs of BPV systems.
photosynthetic cyanobacterium and an engineered exoelectrogenic
Shewanella (Fig. 5C). D-lactate was selected as the energy carrier
responsible for energy transfer between cyanobacteria and Shewanella.
In this microbial consortium, the cyanobacteria capture solar energy and
fix CO2 to synthesize D-lactate, while Shewanella produces electricity by
oxidizing D-lactate, which forms a constrained electron flow from pho­
tons to D-lactate, then to electricity. Through manipulating at the genetic
level, the growth medium level and the device level, the two very
different microorganisms are able to work together happily. The con­
structed BPV system generated a power density of 150 mW/m2 and
maintained stably for over 40 days. In this system, approximately 10.3%
of total fixed light energy was shunted into energy carrier eventually
used for electricity generation. This system demonstrates that a syn­
thetic microbial consortium with constrained electron flow can facilitate
energy transfer from photosynthetic microorganisms to exoelectrogenic
bacteria, achieving higher electron extraction efficiency.
Recently, we reported a novel integrated high-performance BPV
system based on a four-species microbial consortium (Zhu et al., 2022).
This synthetic microbial consortium was composed of an engineered
cyanobacterium, an engineered Escherichia coli, an engineered
S. oneidensis and G. sulfurreducens (Fig. 5D). Among them, the engi­
neered cyanobacterium was responsible for absorbing light energy and
storing the electrons into sucrose, and the engineered E. coli was
responsible for degrading sucrose into lactate. Subsequently, the lactate
was completely oxidized by S. oneidensis and G. sulfurreducens, and the
generated electrons were transferred to the extracellular electrode for
electricity generation. In this four-species BPV system, approximately
70% of total fixed light energy by cyanobacteria was converted into
electricity and the obtained maximum power density was 1700 mW/m2.
Furthermore, a conductive hydrogel was developed to encapsulate
E. coli, S. oneidensis and G. sulfurreducens for preventing them to be
exposed to the aerobic environment resulted by photosynthesis
(Fig. 5D). Finally, the four-species microbial consortium was assembled
into an integrated BPV system, which could directly convert light into
electrical current for over one month, with the maximum energy effi­
ciency of 0.32%. As this integrated BPV system resembles marine mi­
crobial ecosystems in physical structure and ecological structure, it was
also called bionic ocean-battery. In short, these studies demonstrated
that applying synthetic microbial consortia is an effective strategy to
extract photosynthetic electrons for improving power density and pro­
longing the lifetime of BPV systems.
5.3. Synthetic microbial consortia-dependent strategy
As mentioned above, the intrinsic exoelectrogenic activity of
photosynthetic microorganisms is extremely weak. Moreover, the
toxicity and photochemical side reactions of exogenous electron medi­
ators could influence the long-term stability of BPV systems. Therefore,
it is imperative to develop other electron extraction strategies. In
photosynthetic microorganisms, the fixed light energy is preferably
stored into biomass and other organics. Assuming that the electrons
stored in photosynthetic organics could be released efficiently and tar­
geted into electrode, a higher power output of BPV system is expected to
achieve. To this end, a synthetic microbial consortium composed of
photosynthetic microorganisms and exoelectrogenic heterotrophic
bacteria could be considered.
The genera Geobacter and Shewanella are capable of extracting
electrons from organic carbon and converting into electricity through
the relatively efficient EET pathways, thereby suitable to be used to
construct synthetic microbial consortia. Badalamenti et al. (2014) con­
structed a microbial electrochemical cell containing green sulfur bac­
terium Chlorobium and Geobacter. In the light, Chlorobium accumulated
glycogen through photosynthesis. In the dark, Chlorobium ferments
glycogen to acetate, which was subsequently consumed by Geobacter to
produce electric current. A negative light-response of current was
observed in this microbial consortium, due to the acetate was mainly
produced in the dark periods. More importantly, the light-responsive
current was only generated in the co-culture setup, neither monoculture of Chlorobium nor Geobacter, indicating the electric current
was derived from photosynthesis. However, since Chlorobium is an
anoxygenic microorganism, the electrons flowed in this system were
sourced from sulfide rather than from H2O. Moreover, Nishio et al.
(2013) demonstrated that the light-electricity conversion occurred via
syntrophic interactions between algae C. reinhardtii and
G. sulfurreducens, but the oxygen accumulated under light led to the
decrease of electrical current. Liu and Choi (2017b) created a microsized BPV system using a photosynthetic-heterotrophic microbial con­
sortium composed of Synechocystis and S. oneidensis MR-1 (Fig. 5A),
achieving a self-sustaining electricity generation for 13 days. The elec­
tricity was generated by S. oneidensis MR-1 through oxidizing the
organic substrates produced by cyanobacterium. Beyond that, a threespecies microbial consortium composed of two exoelectrogenic bacte­
ria was constructed (Liu et al., 2021). In this consortium, S. oneidensis
MR-1 and Pseudomonas aeruginosa PA01 were simultaneously intro­
duced into system for electricity generation, where the organic com­
pounds were provided as well by Synechocystis through photosynthesis
(Fig. 5B). Moreover, a vertical distribution structure of three species was
created in a solid-state agar-based compartment, which could maximize
the power output and the longevity of the synthetic consortium. Even­
tually, this synthetic three-species consortium generated a maximum
power density of 600 mW/m2, which could power a wireless commu­
nication through series-parallel stacking of fifteen batteries. Since the
photosynthetic microorganisms used in above studies are the wild-types,
only a small amount of organic carbon was released to the outside, and
these photosynthetic products were not fully matched to the substrate
spectrum of exoelectrogenic bacteria. This might restrict the energy flux
from light towards electric current.
To maximally direct photosynthetic electrons towards exoelectro­
genic bacteria, we developed a novel biophotovoltaics system based on a
synthetic microbial consortium with constrained electron flow (Zhu
et al., 2019). This microbial consortium was composed of an engineered
6. Conclusion and perspectives
BPV represents a green power generation technology from sunlight
and water. The major shortcoming of this biohybrid technology is the
inefficient photosynthetic electron transfer across insulating cellular
boundary. To improve electrical outputs, recent studies mainly focused
on abiotic engineering approaches, such as electrode modification, de­
vice miniaturization/optimization, and electron mediator di­
versifications. These efforts are important to improve the performance
of BPV systems from different aspects. Integrating a high-performance
system using these advanced components and designs can be envi­
sioned in the future studies.
The direct electron extraction in single photosynthetic microorgan­
isms relies on intrinsic exoelectrogenic activity or exogenously added
electron mediators. In view of the additional cost and poor photo­
chemical stability, the commonly used exogenous electron mediators
were not suitable to be introduced into BPV systems. Nevertheless,
rational design of other redox molecules targeting to photosystems is
worthy for exploring. Meanwhile, elucidating the exact mechanisms
underlying exoelectrogenic activity of photosynthetic microorganisms is
imperative to understand its physiological significance and genetic
basis. More importantly, it is instructive to targetedly strengthen cor­
responding intrinsic pathways using synthetic biology approaches, such
as overexpression of electron transfer-related proteins, enhancing the de
10
H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
B
A
P. aeruginosa
Synechocystis
S. oneidensis
C
CO2
Electricity
Extracellular electron transfer machinery
Outer
membrane
CBB cycle
Pyruvate
Periplasm
-LDH
hQ
hQ
PSII
4e-
PQH2
Cyt b6f
PSI
ATPase
NAD+
4H++O2
H+
MtrA
MtrC Direct
MtrB
CymA
Acetate+ATP
NapA
PC
2H2O
OmcA
eMQ
2 NADH
CO2
Thylakoid
membrane
Flavin
Nuo
-lactate
NO
H+
2
Electrode
-lactate
NADPH NADP+
H+
ADP
ATP
FNR
H+
e-
Inner
membrane
Flavin
NO3-
Photosynthetic machinery
Synechococcus elongatus UTEX 2973
(Syn2973-omcS-ldh)
Shewanella oneidensis MR-1
(S. oneidensis-∆napA)
D
hQ
O2+4H+
2H2O
Cyanobacteria
O2+4H+
O2+4H+
CO2
2H2O
sucrose
Cyanobacteria
air cathode
Geobacter
CO2
E. coli
CO2
2H2O
acetate
Geobacter
sucrose
CO2
acetate
e-
E. coli
lactate
e-
Shewanella
lactate
Shewanella
Fig. 5. The BPV systems developed using synthetic microbial consortia. (A) A synthetic microbial consortium composed of Synechocystis sp. PCC 6803 and Shewanella
oneidensis MR-1. Reprinted with permission from Journal of Power Sources (Liu and Choi, 2017b). Copyright 2017 Elsevier B.V. (B) A synthetic microbial consortium
composed of Synechocystis sp. PCC 6803, Shewanella oneidensis MR-1 and Pseudomonas aeruginosa PA01. Reprinted with permission from Advanced Energy Materials
(Liu et al., 2021). Copyright 2021 Wiley-VCH GmbH. (C) A synthetic microbial consortium with constrained electron flow, which was composed of an engineered
Synechococcus elongatus UTEX 2973 and an engineered Shewanella oneidensis MR-1. Reprinted with permission from Nature Communications (Zhu et al., 2019).
Copyright 2019 The Authors. (D) A four-species microbial consortium designed for photoelectrical conversion, which was composed of an engineered cyanobac­
terium, an engineered E. coli, an engineered S. oneidensis and G. sulfurreducens (left). This microbial consortium was eventually assembled into a hydrogel-based BPV
system for direct photoelectrical conversion (right). Reprinted with permission from Nature Communications (Zhu et al., 2022). Copyright 2022 The Authors.
11
H. Zhu et al.
Biotechnology Advances 64 (2023) 108101
novo synthesis of endogenous electron mediators, and depriving the
cell-wall components that hinder electron transport. Incorporation of
nanomaterials into photosynthetic microorganisms will also provide
more possibilities in photosynthetic electron extraction (Antonucci
et al., 2022).
The indirect electron extraction using synthetic microbial consortia
shows great potential in power output and long-term running of BPV
systems. In theory, the synthetic microbial consortia can generate
electricity continuously as long as the upstream energy carrier can be
produced continuously by photosynthetic microorganisms, and the
higher the concentration of energy carrier, the greater the power output
will be. At present, the small flux and incapacity of prolonged produc­
tion of energy carriers are the main bottleneck for synthetic consortiadependent BPV systems. Therefore, subsequent studies need to
strengthen the continuous supply of photosynthetic energy carrier by
optimizing the culture conditions or breaking the restrictions caused by
the physiological regulation of photosynthetic microorganisms. More­
over, the quantitative description of the spatial-temporal distribution
patterns for a specific synthetic microbial consortium would be helpful
to elucidate cellular interactions and dynamical changes, and further
guiding to modulate community structure for achieving controllable
outputs.
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CRediT authorship contribution statement
Huawei Zhu: Conceptualization, Investigation, Writing – original
draft, Writing – review & editing, Visualization, Funding acquisition.
Haowei Wang: Investigation, Writing – original draft. Yanping Zhang:
Funding acquisition. Yin Li: Conceptualization, Writing – review &
editing, Funding acquisition.
Declaration of Competing Interest
All authors declare no competing interests.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (32201194, 22072180); the Projects funded by China Post­
doctoral Science Foundation (BX20220333, 2022M710161); the Stra­
tegic Priority Research Program of the Chinese Academy of Sciences
(XDPB18); and the DNL Cooperation Fund, CAS (DNL202014).
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