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Recent advances in biological approaches towards anode biofilm engineering

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 Environ. Eng. Res. 2023; 28(5): 220666 pISSN 1226-1025
https://doi.org/10.4491/eer.2022.666 eISSN 2005-968X

Review

Recent advances in biological approaches towards anode

biofilm engineering for improvement of extracellular electron
transfer in microbial fuel cells
Tahseena Naaz1,*, Ankit Kumar1,*, Anusha Vempaty1,*, Nupur Singhal1, Soumya Pandit1, ,
Pankaj Gautam2, Sokhee P. Jung3,
1
Department of Life Sciences, School of Basic Science and Research, Sharda University, Greater Noida 201306,Uttar Pradesh, India
2
Department of Biotechnology, Graphic Era Deemed to Be University, Dehradun 248002, Uttarakhand, India
3
Department of Environment and Energy Engineering, Chonnam National University, Gwangju 61186, Korea
*
These authors contributed equally to this work.

Received November 12, 2022, Revised January 07, 2023 Accepted January 09, 2023

ABSTRACT
Over the last two decades, scientific communities have been more interested in turning organic waste materials into bioenergy. Microbial
fuel cells (MFC) can degrade organic wastewater and produce electrical power. Many constraints have limited the development of
MFC. Among them, the anode biofilm development is one of the significant constraints that need to be improved. This review delineates
the role of various biological components in the development of electroactive biofilm. The current article focuses on the numerous
electron exchange methods for microbiome-induced electron transfer activity, the different proteins, and secretory chemicals involved
in electron transfer. This study also focuses on several proteomics and genomics methodologies that have been adopted and developed
to improve the extra electron transfer mechanism in electroactive bacteria. Recent advances and publications on synthetic biology and
genetic engineering in investigating the direct and indirect electron transport phenomena have also been highlighted. This review helps
the reader to understand the recent development in the genetic manipulations of the biofilm, electrode material modifications, EET
mechanisms, and operational strategies for improving anode performance. This review also discusses the challenges in present technology
and the future direction for improving biofilm production at the anode.

Keywords: Bioenergy, Biofilm Engineering, Extracellular Electron Transfer, Genetic Engineering, Microbial Fuel Cell, Synthetic Biology

†
This is an Open Access article distributed under the terms Corresponding author
of the Creative Commons Attribution Non-Commercial License E-mail: sokheejung@gmail.com (S.P.J), sounip@gmail.com (S.P)
(http://creativecommons.org/licenses/by-nc/3.0/) which per-
Tel: +82 625301857 (S.P.J.), +91 7044582668 (S.P)
mits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited. Fax:
ORCID: 0000-0002-3566-5649 (S.P.J). 0000-0001-8045-4675 (S.P.)
Copyright © 2023 Korean Society of Environmental Engineers

1
Tahseena Naaz et al.
Graphical Abstract
1. Introduction
Non-renewable energy sources are the most significant contributor
to global energy production because they are potential carriers
of energy that have been generated but cannot be replenished.
Large-scale pollution and climate disruption are typically related
to non-renewable energy utilization. Non-renewable resource deple-
tion became an urgent issue in the 1970s when companies all
over the world experienced abrupt shortages of vital raw materials.
The need for energy around the world is rising fast due to factors
such as population expansion and increased industrial production.
Fossil fuels currently play a significant role in both human existence
and industrial production. The rising global population necessitates
more energy consumption, and the depletion of non-renewable
energy supplies (coal, gas, and oil) poses a danger to the sustain-
ability of the current way of life. One of the most significant obstacles
to human survival and economic growth has been the insufficient
availability of fossil fuels to meet the needs of the ever-increasing
population and increased energy consumption. Problems such as
global warming and pollution increase the need for renewable sour-
ces of energy that can be economical and sustainable. Urbanization,
2
industrialization, depletion of non-renewable resources, and over-
population have all emphasized the need for clean, sustainable,
green, and renewable energy sources. The existence of wind, hydro,
and solar power has satisfied this clause well enough. However,
the equally promising idea of biological fuel cells as a renewable
energy source is still under development and is least explored
[1,2]. In a microbial fuel cell (MFC), the problem of treating waste-
water can be solved by using certain electroactive microbes, which
can perform wastewater treatment and simultaneous bioelectricity
production [3-5]. MFC technology is a step ahead of other traditional
bioenergy production technologies such as anaerobic digestion,
gasification, and fermentation because it significantly reduces sec-
ondary pollutants' levels and is comparatively more cost-effective
[6].
The MFCs comprises two electrodes—cathode and anode- often
separated by a semi-permeable barrier or electrolyte. Electroactive
microbes utilize the anode as a terminal electron acceptor [7].
The anode biofilm degrades organic substrates, produces electrons,
and transports them to the anode surface (Fig. 1) [8]. The irreversible
attachment of electroactive microorganisms to the electrode surface
leads to the growth and development of electroactive biofilm (EAB).

The microorganisms in the EAB use electrodes as electron acceptors
for sustaining their metabolic or respiratory process. Microbial me-
tabolism on the substrates provides energy for the anode biofilm
and the MFC system. In single-chamber cubic MFCs, it takes 9
weeks to establish a stable cell performance, but it takes more
than 17 weeks to get a mature anode biofilm [9]. These biofilms
contain elements that conduct electrons like metal, and their con-
ductivity is affected by various factors such as biofilm thickness,
porosity, microbial composition, microbial viability, etc. Even in
the identical system, the MFC power was significantly different
up to 3.6 times depending on the anode biofilm properties.
The electroactive bacteria transport electron to the electrode
surface via extracellular electron transfer (EET). EET may be done
through artificial mediators (e.g., Methylene blue), natural media-
tors (e.g., Pyocyanin), shuttles produced by the microbes (e.g., C-cy-
tochromes), pili (also known as nanowires), etc [11]. The biofilm
matrix is a heterogeneous medium made up of nucleic acids, poly-
saccharides, lipids, proteins, and so on and is collectively known
as Exopolymeric substances (EPS). EPS provides a microenviron-
ment for EAB for nutrient absorption and assimilation10. In EAB,
EPS also provides an electronic medium for c-cytochromes and
pili, which help discharge electrons and their transportation
[9,11,12].
MFC’s mechanism deals with two types of microorganisms. One
of them is electroactive microorganisms (EAMs), also known as
exoelectrogens, electrogens, electricegens, exoelectrogenic bacteria,
or anode-respiring bacteria, which oxidize organic material and
release electrons onto the electrode [13]. The other type is non-EAMs,
often referred to as endo-electrogens or non-exoelectrogens, because
they either consume electrons or produce mediators that direct
the electron route [14,15]. These non-EAMs can swim freely in
the water as planktonic growth or stick together like a blanket
in the EAB along with old cells [16]. When cells contact a solid
and hard surface, they undergo a series of anatomical, biochemical,
and physiological adaptations that mark the shift of planktonic
growth into a sessile state. Non-EAMs are important in the MFC
ecological system because they help produce dense biofilm and
maintain an anaerobic condition by utilizing oxygen that would
otherwise pollute the anode [13]. Compared to MFCs containing
pure exoelectrogens, MFCs with mixed consortia exhibit increased
power, durability, and the capacity to break down complex com-
pounds and convert them into electricity [17].
It is possible to classify the most common uses of MFCs that
have emerged in the past few decades as follows:
• Electricity generation: MFCs are designed to provide adequate
power for low-powered electronic gadgets. Two days of oper-
ation of ten LED lights and one digital clock using a stackable
MFC that Rahimnejad et al. depicted were successful [18].
• Biohydrogen production: Simple modifications allow MFCs
to collect biohydrogen instead of electricity generation. The
hydrogen can be stored for future use [19]. MFCs provide
a renewable hydrogen source in a hydrogen economy that
can contribute to the total hydrogen demand [20,21].
• Wastewater treatment: Wastewaters from a variety of in-
dustries, including sewage, the food processing industry,
wastewater of swine, and the corn ethanol industry, all con-
3
Environmental Engineering Research 28(5) 220666
stitute biodegradable organic materials that can be converted
into energy [19,22]. Long-detention-time anaerobic digestion
processes are best suited for high-strength wastewater because
they produce electricity and methane simultaneously from
waste materials [23]. There have been reports of columbic
efficiencies of up to 80% [24,25], allowing for the removal
of up to 90% of the COD [26].
• MFCs in biosensors: MFCs are convenient for fuelling electro-
chemical sensors and are compact telemetry systems for trans-
mitting data to distant receivers due to the batteries' limited
lifetime and recharging requirements [27,28]. Using MFCs as
a sensor for biological oxygen demand (BOD) has been shown
to work [28]. This kind of sensor for BOD has excellent reprodu-
cibility and operational sustainability and can be kept running
for 5 years [29].
Though the MFC technology has several advantages, its commer-
cialization is yet to be seen. Therefore, it is critical to identify
the flaws that impede MFC performance and its various applications.
The role of biofilms in electricity production is necessary to under-
stand the factors affecting biofilm production and the mechanism
of EET [30]. While most studies focus on electron transfer techni-
ques, the critical research questions for MFCs concern topics such
as (1) redesigning exoelectrogens to make them more efficient and
(2) developing inexpensive and highly efficient materials that can
be used for power production and sewage treatment; 3) learning
how to promote biofilm development in an MFC to enhance energy
output; and 4) need to gain insight into a wide range of EAB phenom-
ena, remained unanswered.
Biofilm-coated electrodes for bioelectricity production via MFCs
have sparked much attention in the scientific community. Various
experts recommend thick anode biofilm formation owes to the
synergistic influence of microbial communities [17]. Even though
MFC technology seems to be a partial solution to the current energy
crisis, it is impossible not to view the MFCs as a sustainable energy
source that can consistently power contemporary society while
simultaneously treating wastewater [31]. Realizing the potential
for extensive conversion of organic waste and biomass into bio-
energy, this technology can be used to develop solutions for recharg-
ing biomedical devices, running home appliances, and other elec-
trical devices [32]. However, only a handful of literature is presently
discussing the challenges that affect the anode biofilm formation,
which is one of the significant limitations of electricity production
in MFCs. There is a need for a detailed discussion of the different
strategies used to enhance biofilm formation and its future research
direction. This review article aims to paint a detailed picture of
the development of the EAB and the challenges involved, and
the various strategies used to overcome the challenges. The present
review focuses on the development of EAB and the microorganisms
responsible. It will also focus on the various factors affecting biofilm
formation, the hypothesized mechanisms by which the electrons
are transported through the biofilm, the challenges that limit the
electricity production by the biofilm, and the most recent advances
in the strategies used to promote biofilm formation for enhanced
electricity production. The present study will also discuss the future
perspective of anode biofilm development for increased electricity
production.
Tahseena Naaz et al.
2. Microorganisms Generating Power in MFCs
The majority of MFCs generate electricity from sewage. Sediments
occurring in open systems are prone to the occurrence of electrogenic
community selection. Communities of electron-transferring micro-
organisms at the anode chamber's electrode should perform actions
similar to anaerobic digester methanogenic communities [33]. These
microbiocenoses are known as anodophilic [34]. There are typically
delta-Proteobacteria at the anode of sedimentary systems, ranging
from 50% to 90%. Gamma-Proteobacteria (9%), Firmicutes (11.6%),
and Cytophagales (33%) are also present, however, to a lesser extent
[35]. Because of the unique abilities to generate EAB and highly
efficient EPS, many photosynthetic and anaerobic biological mi-
crobes have been utilized in MFCs as donors and recipients of
electrons, which include Saccharomyces cerevisiae [36],
Rhodispirullum rubrum [37], Chlorella vulgaris [38], Thiobacillus
ferrooxidance, Phormidium sp. [39], Leptothrix discophora [40],
Desulfovibrio desulfuricans [41], Pseudomonas aeroginosa [42],
Klebsiellapneumoniae [43], Geobacter metallireducens [14],
Shewanella oneidensis along with a few other anaerobic microbes.
The genetics of certain wild-type strains have been modified using
recombinant DNA technology to improve current production and
sustained biomass generation.
3. Types of EET
3.1. Direct Electron Transfer (DET)
Certain electroactive bacteria, especially Shewanella oneidensis,
Rhodoferax ferrireducens and Geobacter sulfurreducens, use direct
electron transfer (DET) for the transfer of electrons to the electrode
surface via redox-active proteins or conductive pili (also known
as nanowires). For DET, the microbes are closely associated with
the electrode because only immobilized cells can effectively perform
DET. This leads to a high rate of transfer of electrons from the
microbes to the electrode, resulting in high power production.
These bacteria used transmembrane proteins such as cytochrome
c found on the cell wall to transfer electrons from their cell walls
to the electrodes. The cell wall of the bacteria, or maybe the mem-
branes of the organelle, must be directly connected to the electrodes
of the MFC for DET to occur [44]. Exoelectrogens transport electrons
to the anode via the help of suitable electron shuttles in two different
methods: (i) the transfer of electrons across short distances by
redox-active proteins on the cell wall of bacteria, such as cyto-
chromes, and (ii) nanowires, or conducting pili, allow for electron
Table 1. The diversification of mechanisms in the transfer of electrons by various types of electrogenic microbes in Microbial Fuel Cells.
Domain Species Mode of Electron Transfer at Anode Mode of Electron Transfer at Cathode Reference
Fungi Saccharomyces cerevisiae Mediated ET
Bacteria Geobacter sulfureducens Direct ET
Sporomusa ovata Not yet confirmed
Clostridium lijungdahlii Not yet confirmed
Shewanella oneidensis Direct ET
4
transport across long distances [30]. Live cells are often assumed
to remain electrically dormant owing to their non-conductive prop-
erties [45]. However, studies have shown that it's possible to make
good use of an organism inside an EAB since it has an electron
transfer protein (ETP) bound by membranes [46]. Additionally,
as shown by the literature, carrier proteins direct the transfer of
electrons from the interior to the exterior of the cell. Redox proteins
in the outer membrane, however, often carry external electrons
to a solid final electron acceptor, such as an electrode [47]. Geobacter
sp. [48], Rhodoferax sp. [49], and Shewanella sp. [50] have been
reported to have multi-heme protein molecules along with c-type
cytochromes and are also found with EAB [30]. In exoelectrogens,
the flavins, as well as cytochromes, are found to be interconnected
cyclic compounds with a strong redox capacity and are electron
transporters. Shewanella oneidensis was shown to actively partic-
ipate in electron transfer through electron shuttles and metallic
reduction based on cyclic voltammetry (CV) study of the redox
properties of flavins like FAD and vitamin B2 [51]. A hybrid strain
MR-1 of Shewanella oneidensis, in which CymA is abundantly
expressed, was also studied for its possible application in MFCs
[52]. Recent research has also shown that Bacillus megaterium
relies on flavins for electron transfer, joining the ranks of well-known
flavin producers like Shewanella oneidensis [53]. Cytochromes are
mainly proteins containing heme that shuttle electrons across the
membrane and into the surrounding medium. Several studies have
reported the presence of electron mediators in the EPS matrix
of biofilms [54], the activity of outer membrane cytochromes in
electrode and metal reduction, and the function of pili/nanowire
in biofilm development [55].
In addition to cytochromes, bacteria have sticky pili that are
mostly made of sortase enzymes and proteins [56]. According to
earlier research, several Geobacter and Shewanella strains develop
electron-conducting pili. These pili permit such organisms to the
long-range transfer of electrons to the electrode that is not in immedi-
ate contact with the microbes. These nanowires, or pili, attach
to the cytochromes of cells and carry electrons from the outer
membrane to the electrode [57]. These pili (nanowires) even promote
the formation of thick EAB, further improving MFC performance
[30]. Some of the microbes and their chosen methods of electron
transfer are listed in Table 1.
3.2. Indirect Electron Transfer
Naturally produced redox mediators, known as secondary metabo-
lites, are relevant in Indirect Electron Transfer (IDET) or mediated
electron transfer (MET) [58]. In IDET, the synthesis of naturally
produced mediators renders electron transfer without needing redox
electron shuttles from the outside [59].
MET (Mediated Electron Transfer) [193]
Direct ET [194]
H2 mediated ET [195]
Direct ET (hypothetically) [196]
Mediated ET [197]

This can also be linked to soluble electron transporters released
by microbes such as flavins and pyocyanin. The mediator accepts
and donates electrons from the bacterial cell to a strong oxidant
(anode) or into anaerobic biofilm sheets. They are reoxidized and
made accessible for further redox activities. As a result, a molecule
may support hundreds of redox cycles [60]. Thus, the micro-
organisms can dispose of electrons at increasing speed levels by
making minute amounts of such compounds [44]. P. aeruginosa
produces a pigment, Pyocyanin, that has previously been identified
as required for conducting IDET in bacteria [61]. Shewanella onei-
densis produced a mediator known as quinone. The quinone-treated
version of MFC has shown double power output compared to the
mediator-free version [61]. Pseudomonas alcaliphila can also pro-
duce redox mediators through bacterial synthesis. Thus, in addition
to redox mediators, the oxidation of the metabolites also contributes
to IDET. Bacterial fermentation causes the anode to produce hydro-
gen when oxidized [62]. Immediate bacterial interaction with the
anode or soluble redox active mediators such as pigments, ubiq-
uinone, metal complexes, and dyes, and readily soluble electron
carriers are also viable options for transferring electrons from the
electrode to the bacteria [44].
4. Factors Affecting the Formation of Biofilm
Extracellular polymeric substances (EPS) and lipopolysaccharides
(LPS) play a crucial role in biofilm development alongside exterior
environmental circumstances and genomic pathways that ulti-
mately result in biofilm formation in cells [63]. Signaling molecules
of quorum sensing (QS) also control biofilm formation [64]. High
temperatures, presence of heavy metals [65], salinity [66], pH [67],
nutrient starvation [58], nutrient depletion, the flow rate of moving
water bodies, growth substrate, and so on are some of the significant
factors that affect the synthesis of EPS and signaling molecules,
thus affecting the biofilm formation [68]. While genetically en-
gineered molecules may have the upper hand over environmental
factors, while forming biofilms, EPS and quorum-sensing molecules
cannot be excluded from the limelight [30]. But further research
into this domain is required for better understanding.
4.1. Role of Exopolysaccharides (EPS) and Lipopoly-
saccharides (LPS) in Electron Transfer
Changes in EPS contents seem to have significant ramifications
in terms of surface energy and external adhesion, providing a founda-
tion for preserving accessory peptides implicated in intercellular
interaction, as seen in Shewanella sp [30]. In this regard, studies
with Geobacter sulfurreducens have drawn attention, revealing the
role of EPS as adhesive sites for exoelectrogens, allowing them
to transport electrons. Results show that the formation of ion-con-
ducting biofilms on the cathode and anode was absent in EPS-defi-
cient mutants. Geobacter sulfurreducens, under standard con-
ditions, produce peripheral anchoring polymers, which are required
for electron transfer to the electrode and include c-type cytochrome
sites [69].
EPS includes polysaccharides that improve biofilm formation,
such as Alginate and Pel, which are responsible for generating
5
Environmental Engineering Research 28(5) 220666
high levels of c-di-GMP by diguanylate cyclases. Psl (a type of
polysaccharide) is responsible for initiating and maintaining the
biofilm structure [70]. At the same time, Pel, a matrix rich in glucose,
helps biofilm formation associated with the solid surface for
non-pileated organisms [71]. For example, in Pseudomonas aerugi-
nosa, the algC gene produces an enzyme essential for the production
of polysaccharides, namely, Psl, LPS, alginate, and Pel, which influ-
ence biofilm formation [72]. As mentioned earlier, most of the
genes that make any of the polysaccharides are typically involved
in producing biofilms [72].
A thorough examination of the microbial community in electro-
genic multilayered populations provides information on the proc-
esses that convert complex compounds (organic) in industrial efflu-
ent to electric power in MFCs [73]. Direct interspecies electron
transfer (DIET), occurring across bacteria or in combination with
electromechanical particles, can be implemented in designed organ-
izations to improve sewage treatment goals and for regenerative
braking in bio-electrochemical advances [74]. As a result, EPS’s
role in biofilm generated by microbes in MFCs has become a focus
for scientists.
4.2. Cyclic di-GMP Signaling
C-di-GMP is a signaling component of a molecule that coordinates
behavioral change from mobility to sessility and vice-versa (also
called dispersion) [75]. Several bacteria, including Serovar typhimu-
rium, Salmonella enterica, Pseudomonas aeruginosa, and
Escherichia coli, have shown a connection between higher c-di-GMP
concentrations in organism and biofilm generation or lower
c-di-GMP concentrations and mobility [76]. The cyclic di-GMP
controls biofilm production and dispersal via many genetic alter-
ations [77]. Researchers report that MucR (membrane-associated
regulator of alginate biosynthesis in biofilms, acts as
Phosphodiesterase (PDE) and a positive controller of the dispersal
of biofilms triggered by nitric oxide or glutamate [78].
Diguanylate cyclases (DGCs) and PDEs have been recognized
as key players in the biofilm development of bacteria such as
Pseudomonas aeruginosa. A thorough analysis of the characteristics
of PDE and DGC mutants, together with epistasis research, revealed
details regarding the participation of BifA (PDE) or RoeA (DGC)
and SadC (DGC). As a result, there is a greater understanding of
their perceived importance at various stages of the biofilm formation
process [77]. In the given Fig. 1 (a) and (b), we demonstrate this
notion developed by combining all the Pseudomonas aeruginosa’s
PDEs and DGCs that have been found that take part in biofilm
development. WspR, SadC, RoeA, SiaD, and YfiN/TpbB are the
DGCs that are identified to be controlling the shift from planktonic
growth to sessile cell growth of the bacteria, enhancing the biofilm
formation [79]. The DGCs – NicD and GcbA, as well as the PDEs
RbdA, NbdA, and DipA (Pch), are associated with the growth of
the biofilm as represented diagrammatically in Fig. 1(c) [80].
4.3. Quorum Sensing
The microbial population interacts among itself via cell-to-cell asso-
ciation, coordinating their activities. Hence, this necessitates the
generation of signaling molecules from the colonies, which results
in the phenomena known as quorum sensing [30]. Quorum Sensing
Tahseena Naaz et al.

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 Environmental Engineering Research 28(5) 220666

Fig 1. Mechanisms of cyclic di-GMP signaling pathways (a) The relationship of C-di GMP signaling with DGC and QS; (b) Biochemical pathway
for Geobacter sp. and Shewanella sp. carrying out Cyclic di-GMP signaling; (c) Regulation of P. aeruginosa biofilm growth by c-di-GMP
signal transduction pathways via two network cascades. The upper section depicts PDE, DGC, and c-di-GMP receptors and the developmental
period during which these are thought to function. The bottom section depicts two phase-specific different regulation mechanisms for
biofilms. The range of yellow panels in the figure backdrop reflects growing internalized c-di-GMP concentrations (denoted by pink *
with increasing amounts).

(QS) signaling molecules differ between Gram +ve and Gram -ve
bacteria, as given in Table 2. The first question that arises when
considering the relation between biofilm growth and QS is when
the microbial density exceeds the critical level that permits QS
signaling to engage in biofilm management. Because it restricts
microbes that are moving freely in the medium, the preliminary
adherence phase appears to be suboptimal for collecting QS. Later,
whenever the attached microbes split and form microcolonies, the
total population rises, leading to the QS reaching adequate levels
to regulate the development and breakdown of the biofilm. Recent
studies have shown that many bacterial species use QS to coordinate
the dispersal of the biofilm. Dispersion of biofilms is required for
bacteria to grow [81]. When nutrients and other elements become
scarce and waste piles up, biofilm dispersal is critical for bacteria
to break free and colonize habitats. Increasing the quantities of
QS signals further may cause malnutrition and stress in the plank-
tonic bacterium population, creating an unfavorable environment.
Bacteria use biofilms to protect themselves from this stress. The
environment on the biofilm's surface is favorable for microbe growth
[82].
In general, there are three kinds of QS systems: (1) AHL
(Acylhomoserine lactones) are employed as signal molecules in
gram-negative microorganisms, (2) oligopeptides are QS molecules
generally found in gram-positive microorganisms, and (3) luxS-en-
coded autoinducers QS 2 (AI-2) are found in Gram -ve as well
as Gram +ve microbes [83]. EPS production, chemotactic processes,
and cell migration play an important role during the degradation
of organic pollutants or purification. It is found that gene mod-
ification of QS may assist in creating designed biofilms with en-
hanced degradation kinetic models and increase the performance
of MFCs [84].
Moreover, a rhamnolipid synthesis which QS controls contrib-
utes to biofilm design by maintaining open biofilm water channels
during matrix growth [85]. Several studies indicated that rhlL a
rhamnolipid synthesis gene, also regulates the production of secon-
dary metabolites such as pyocyanin in P. aeruginosa [86]. It is
still uncertain if population size affects the production of one of
these polymers. As a result, the molecular pathways that explain
the connection between QS and biofilm formation in Pseudomonas
aeruginosa remain unclear. It is also found that Vibrio cholerae
starts producing EPS even when in a low-density population, prob-
ably before even forming a heterogenous community. The QS com-
pounds and the taxa that employ them are presented in Table 2.

7
Tahseena Naaz et al.

Table 2. Various QS agents and their respective species associated with them are listed below.
Species of microbes QS agents as chemicals Reference
Xylella fastidosa, Xanthomonas (Gram -ve) DSF [198]
Streptomyces spp. (Gram +ve) Butyrolactones [199]
Pseudomonas fluorescens, pseudomonas syringae AHL [200]
Gram +ve bacteria (in general) Oligopeptides [201]

Table 3. List of biosurfactant substances, the parameters they work under, and the impact they have on MFC and currents output
Parameters Biosurfactant materials Impact on the system Reference
Phenazine Generation Sophorolipids 1.7 times lesser [202]
Biodegradation Tween -80 43.5% higher [203]
Power Trehalose 5.93 times higher [204]
SDS 20% higher [119]
Sophorolipids 2.6-4% higher [202]
Tween-80 88% higher [205]
Current Rhamnolipid Synergistic effect on extracellular transfer of electrons [206]
Trehalose 1.83 times higher [207]
Biofilm intensity Rhamnolipid 2 times thicker [208]
Hydrogen generation Rhamnolipid 4 times higher [209]
Internal resistance Trehalose 43% lower [204]
Sophorolipid 40% lower [17]
Rhamnolipid 30% lower [210]

Some anaerobic, facultative microorganisms, such as
Pseudomonas aeruginosa along with Salmonella enteritidis, rely
heavily on QS for their growth and biofilm formation. QS regulates
the anaerobic growth of Pseudomonas aeruginosa PAO1, where
the outer sheath proteins are Rhl and OprF. QS pathways have
been identified to be essential for successful bacterial biofilm for-
mation [87]. According to reports, bacterial growth is significantly
reduced when OprF is not functional. However, a lacking rhlR
or, sometimes, rhlurel leads microbes to undergo apoptosis owing
to increased nitric oxide production. Moreover, the gene expression
of Pseudomonas aeruginosa is usually regulated by AHL signals
dependent on cell density, while QS in Salmonella enteritidis is
mediated by signaling molecules AI-2, AI-1, and AI-3 [88].
4.4. Effect of Biosurfactants on Biofilm Formation
Biosurfactants are a type of surfactant that is produced by bacteria
or fungi and have intense interfacial activity, emulsifying action,
and foaming properties. Biosurfactants can change the ultra-
structures of the cell membranes to form transmembrane channels,
which are effective for enhancing cell permeability, reducing mem-
brane resistance, and increasing the electron transport of the mi-
crobes in the EAB. It leads to an increase in substrate degradation,
increasing the bioavailability of nutrients. According to recent stud-
ies, applying biosurfactants promotes microbial degradation of
crude oil in MFCs, which may impact the taxonomic composition
of EAB [89]. The researchers studied five detergents and discovered
that the lecithosampholytic surfactants (primarily lecithin) ex-
hibited high power output and high microbial degradation. The
most selective alterations in bacterial populations were produced
by Sodium dodecyl sulfate (SDS) and -cyclodextrin (biosurfactant).
Another research found that the inclusion of Tween [80] improved
polychlorinated biphenyl degradation in MFC by 43.5% [90]. The
hydrophobic substrate's bioavailability increases with the presence
of biosurfactants [91]. Both biotic and abiotic surfaces can benefit
from film conditioning when biosurfactants are released onto them
[92]. The hydrophobicity of the cell surface increases with bio-
surfactants like rhamnolipids. Because of their ability to condition
films and modify the hydrophobicity of bacterial surfaces, bio-
surfactants facilitate the adhesion of bacteria to surfaces, a necessary
step in the development of biofilms [93]. Lipopeptide biosynthesis
has been shown to reduce the hydrophobicity of cell surfaces and
the Bacillus spp.’s adhesion to the stainless steel surface [94].
Biosurfactants were also shown to play a crucial role in the biofilm
formation and adsorption of S. epidermidis, S. aureus, and P. aerugi-
nosa at the -interface of ƞ-decane–water in another study [95].
Some of the surfactants and their impact on the EET of the micro-
organisms are given in Table 3.
Only rhamnolipids and sophorolipids were discovered as bio-
logical detergents, even after multiple trials with various detergents.
Reports state that biosurfactants may interact with electron transport
channels such as phenazines, biofilm adherence structures, and
survivability at the electrode. Adding biosurfactants and detergents

8

Table 4. Various genetic modification techniques to enhance the functioning of MFC by improvising the mechanism of EET.
Microbial species Genetic modification
Shewanella oneidensis Excessive expression of D-lactate transporter
Escherichia coli Expression of CymA and MtrCAB from S. oneidensis
Pseudomonas aeruginosa Expression of IrrE from Deinococcus radiodurans
Synechococcus elongatus Expression of OmcS from G. sulfurreducens
Geobacter sulfurreducens Excision of GSU1240
Shewanella oneidensis Suppression of UvrY and expression of SpeF via CRISPRi 1.7x increase in current
Escherichia coli Omission of ldhA
in MFCs would've had the primary advantage of assuring an en-
ergy-neutral change, positively enhancing MFC’s performance in
substrate utilization and energy output via multiple pathways.
4.5. External Resistance
External resistance (Rext) in an MFC regulates the anode availability
to the exoelectrogens as an electron acceptor. It affects anode poten-
tial, EET rate, current production, and anodic microbial diversity.
A lower Rext application increases electrode potential, EET rate,
current production and microbial diversity in the anode [16]. It
was reported that it's not only electrochemistry hat gets affected
by external resistance but the relative diversity of the microbial
community also, as shown by analysis of ribosomal intergenic
space [96]. Optimum external resistance, suggested by Jung’s group
[97–99], is the external resistance at which an MFC produces its
maximum power density according to the electric power relation-
ship (P = IV). The maximum power point tracking method is also
conducted by this algorithm that finds the external resistor that
produces the maximum power.
When an anode biofilm adapts to its optimum external resistance
in each stage, MFC power production gradually increases as the
operation continues and reaches a stable performance [9]. Therefore,
operating an MFC at its optimum external resistance is very
important. Since optimal external resistance varies depending on
cell conditions such as anode biofilm, MFC structure, medium
properties, substrate concentration, etc., it is necessary to measure
and apply optimal external resistance periodically. An optimum
resistance value can be obtained by a polarization curve or an
impedance test. The optimum external resistance is generally equal
to the internal resistance of the system. When the external resistance
is higher or lower than the optimal external resistance, the produced
power will be lower than the maximum power.
For wastewater treatment, a lower external resistance can boost
wastewater treatment. The low MFC power due to low external
resistance application can be solved by utilizing a power-boosting
electrical circuit.
4.6. Microbial Cooperation and Competition
Bacterial species have been found to have a wide variety of strategies
for coexisting with or dominating other microbes competing over
the same resources, as revealed by studies of interspecies com-
petitive strategies [100]. Bacteria not only compete with one another
within their species, but they can also participate in cooperative
activities [101]. When present in mixed populations, these intra-
9
Environmental Engineering Research 28(5) 220666
Performance Reference
1.3x increase in current [211]
4x increase in power [212]
+71% current [213]
9x increase in current [214]
+50% current [215]
[216]
6x increase in power [217]
specific processes can potentially affect the communication be-
tween species, such as by facilitating better strategies for competing
[102]. In a growth environment with low nutrients level, the mem-
bers of the mixed culture will compete for the same resource because
of the depletion of resources due to a continuous increase in the
population [103]. When there are several different ecological niches
available, as in a biofilm or a static liquid culture, natural selection
will favor the variants that are best able to colonize the various
niches [104]. This antagonistic behavior in mixed culture can be
tackled by selecting different organisms with different substrate
requirements. This method of co-culturing has resulted in excellent
outcomes in the degradation of waste materials and drug delivery
[105].
5. Genetic Modifications
Genetically modifying the exoelectrogens can be done through sev-
eral methods, such as metabolic engineering. Studies have, up
to this point, mainly concentrated on the alteration of the cell
membrane and the overexpression of redox mediators or elec-
tron-conducting proteins. Different genetic modification techniques
to increase the performance of an MFC by improving EET can
be seen in Table 4.
5.1. Genetic Modifications on Cytochrome to Improve Biofilm
Production
In Shewanella oneidensis, EET transfer is done through metal re-
ducing pathway consisting of cytochromes (Fcc, MtrB, OmcA, MtrF,
MtrA, CymA, MtrC, etc.). Still, in Geobacter, the attached PCC
(porin-cytochrome combination) in the outer surface helps in the
EET [106]. Cytochromes are cell-bound multiheme-binding im-
portant for the DIET and oxidative phosphorylation. The heme
group and the adaptable cytochrome-c [107] perform critical func-
tions in the electron transport process. A CV study of c-type cyto-
chrome electrochemical activity in Shewanella lochia PV-4 pro-
vided us with the redox maxima induced by the exchange of elec-
trons between the electrodes and active redox complex on bacteria’s
cellular membranes. Both cathodic and anodic fluxes in the multi-
layered biofilm were more significant than those in the single-layer
biofilm due to the enhanced transfer of electrons caused by more
c-type cytochromes on the cell’s surface and matrix. Other studies
show that a cytochrome in Shewanella oneidensis is transcribed
by the OmcA gene. S. oneidensis, in a mutant devoid of the same
Tahseena Naaz et al.

OmcA, generated 40% less current [108]. In G. sulfurreducens,
OmcZ encodes a cytochrome that helps in biofilm growth and
is essential for electron transfer. The OmcZ gene deficiency reduced
electrochemical activity compared to the wild type. OmcZ works
as an electrochemical gateway, allowing electrons to travel from
the biofilm to the electrode [109]. The most recent studies discovered
that overexpression of CymA in S. oneidensis MR-1 had a greater
power output and specific growth rate in an MFC over wild-type
MR-1 [110].
5.2. Overexpression of Electron-Conducting Proteins
A study investigated the role of the PilA structural protein, linked
to pilus formation, in altering the conductivity of G. sulfurreducens
biofilms. This was done assuming that pili-based nanowires make
up a significant component of the EET of G. sulfurreducens. The
expression of PilA was shown to correlate with the filaments' elec-
tron conductivity in the wild-type biofilm. PilA-deficient mutants
exhibited decreased conductivity, confirming the role of the pilA
gene in developing conducting biofilm networks and laying the
groundwork for future genetic improvements [111]. Studies were
carried out with mutants showing deletions in the regulatory pilZ
gene to assess the favorable impact of PilA overexpression on the
conductivity of G. sulfurreducens biofilms. This deletion produced
noticeably more cohesive biofilms with higher conductivity in one
of the mutants and enhanced PilA expression [112]. These findings
show that a controlled increase in the number of nanowires within
a biofilm that is electrochemically active can be a helpful technique
for increasing the EET of EAB [113]. The question of whether
c-type cytochromes, including OmcS filaments, or solely pili-based
filaments, are in charge of long-distance electron transport is still
up for debate. The overexpression of redox mediators like pyocyanin
by P. aeruginosa can enhance the EET in addition to nanowires.
P. aeruginosa's biosynthesis pathway was genetically altered, lead-
ing to an increase in power density by four times and a 1.6-fold
increase in pyocyanin concentration [114]. Additionally, over-
expressing NAD synthase in P. aeruginosa increased the quantity
of intracellular NAD+/NADH readily available, which boosted the
production of pyocyanin by a factor of 1.5 and the power density
by a factor of 3 [115].
5.3. Manipulation of the Cell Envelope
The impact of cell secretions and their overexpression of redox
mediators on the EET pathways was studied. A study exposed
P. aeruginosa to common membrane-disintegrating agents like
EDTA (ethylenediamine tetra acetic acid) or surfactants and tested
the membrane permeability of P. aeruginosa. It led to increased
membrane permeability, leading to high power production in MFC.
Similar experiments conducted by modifying sophorolipids
(bio-surfactant) showed similar results in MFC. The rhlA gene,
which is in charge of P. aeruginosa’s rhamnolipid synthesis, was
overexpressed to avoid external surfactant addition, boosting the
membrane permeability to pyocyanin and the power density and
by 2.5-fold in contrast to the control. A transposon could be inserted
into the S. oneidensis MR-1 polysaccharide biosynthesis gene cluster
to affect the cell membrane. This method produced a power density
that was 50% higher and a less noticeable polysaccharide capsule.
A better cell adherence to the supplied graphite anode and a decrease
in the development of non-conducting surface structures were found
to be the causes of this variation [116].
6. Modification of Electrodes to Promote Anodic
Biofilm Formation: A Strategy for Modifying
Various Types of Electrodes.
Electrocatalysts are often immobilized on electrodes used in MFCs;
however, in other cases, the material of the electrodes acts as a
catalyst. These electrodes should comprise a wide surface area,
low resistance, high conductivity, along with low impedance.
Furthermore, the electrode material should be biocompatible, allow-
ing for the growth and adhesion of microorganisms on the surface
of the electrode [117]. But somehow, some materials may fail to
mediate electron transport and biofilm formation. These electrodes
must be modified to promote EET and biofilm formation using
various methods to enhance power production in MFC. The micro-
environment produced by the coexistence of numerous bacterial
species present in the biofilm drives its metabolism [97, 98].
Acidogenic and fermentative bacteria interact to break down com-
plex organic molecules into molecular hydrogen. A perfect habitat
for developing microbial symbiotic relationships is the micro-
environmental niche found inside biofilms.
Moreover, interactions between populations of metabolically co-
operative microbes support the growth of colonies inside the biofilm.
One or more microbial species may build colonization depending
on the food and metabolite exchanges. Several anode electrode
modifications were studied in MFC. Carbon nanotubes (CNT) have
been used as a promising electrode material. The CNT is a carbon
electrode material with a vast surface area that is formed like a
cylinder. It also has excellent electrical conductivity, is physically
stable, and is chemically inert. Researchers created a modified
anode composed of Carbon cloth (CC) that was embellished with
nitrogen-doped CNT (NCNT) that resembles bamboo to increase
the biocompatibility of the CNT to encourage microbial attach-
ment111. Compared to unmodified ones, the bamboo-like NCNT
considerably improved MFC performance and decreased the in-
ternal resistance of the anode. It is evident that modifying the
anode electrode with bamboo-like NCNT considerably increased
the anodic potential and current density. This was attributed to
the increased number of active sites brought on by various joint
structures as well as the nitrogen dopant’s improved
biocompatibility. Ohmic and non-ohmic losses have been linked
to high internal resistance. By simply covering the electrode with
a biofilm, it is possible to reduce the distance between the bacterial
cells and the electrode and therefore reduce the charge transfer
loss.
To lower the MFC's operational costs, metal was used as the
electrode material. They are reported to be corrosion-resistant and
reasonably affordable. Metals like stainless steel fiber felt (SSFF),
and stainless-steel mesh (SSM) have been used to operate as the
MFC’s anodic current collector and as a 3D support, respectively
[118]. However, the metal-based fiber has muscular strength, con-

10

Table 5. Electrode modifications to enhance MFC performance.
Bacteria/biocatalyst Anode material
source
Aerobic & anaerobic Bamboo-NCNT
sludge
Geobacter sp. SS-Felt
Shewanella Carbon coated Fe2O3
oneidensis
Escherichia coli Polyaniline
Shewanella putrefaciens graphene
ductivity, and corrosion resistance; its low biocompatibility and
significant over-potential limit its applicability in more MFC
applications. Therefore, using binder-assisted pasting, the perform-
ance of SSFF was enhanced through modification with either gra-
phene, CNT, or activated carbon. When MFC is used with SSM
as the anode, Song et al. found that the start-up time and activation
resistance are reduced. Screen printing sodium dodecyl sulfate
(SDS) composited with exfoliated graphene-multiwalled CNT onto
the SSM was used to modify the electrode. Their research revealed
that the redesigned anode had better hydrophilicity and bio-
compatibility [119].
Due to a reduction in ohmic loss resulting from improved bacterial
cell adherence, electrode modification with nanocomposite metal
or its oxides considerably improved MFC performance. Along with
improving long-range EET in MFC, metal oxides like goethite and
rutile also promote the growth of chemotrophic and heterotrophic
bacteria by harnessing solar energy. The carbonaceous material
has been combined with the oxides of titanium (Ti), tin (Sn), man-
ganese (Mn), iron (Fe), and other metals to create nanocomposite,
which has been used as an anode electrode modifier.
Conductive polymers have gained popularity as anode doping
materials in MFC because of their conductivity. They are usually
used to modify anode electrodes to improve the biocompatibility
of the anodes. When the polymer is doped with a nanomaterial
in the form of composites, enhanced anodic performance is reported.
To increase bacterial cell adhesion and promote EET of EAB, certain
functional groups are introduced onto the anode surfaces via electro-
chemical oxidation, a quick and inexpensive way of anode material
modification. The technique calls for a low treatment time and
ambient temperature. For electrolysis-based surface modification
of the carbon cloth anode, three alternative electrolytes have been
investigated. The adjustment resulted in a quicker start-up time
while increasing the anode electrode’s wettability parameter and
electrochemically accessible surface area. However, this benefit
gradually disappears with time, most likely due to the declining
metabolic activity of the biomass adhering to the electrode surface
[120]. It has been demonstrated that amide groups added to the
surface of activated carbon felt (ACF) modified with ethylenedi-
amine promote EET and increase microbial cell adhesion more
than unmodified ACF changed with nitric acid. Although the
start-up time of MFC was sped up by both techniques, compared
to unmodified ACF, the amide group was more biocompatible in
modified ACF with ethylenediamine. Compared to pristine carbon
paper (CP), the MFC operated with CP/PANI electrode modified
Environmental Engineering Research 28(5) 220666
Electrode size Performance Reference
4.5 cm2 1040 mW/m2 [218]
2×1×0.07 cm3 27.42 ± 1.14 mA/cm3 [219]
- 0.22 ± 0.01 mA/m2 [220]
1.5 cm2 2059 mW/m2 [221]
2 cm2 3903 mW/m2 [222]
with HNO3, ethylenediamine, and diethanolamine used less sub-
strate to reach greater power densities. Due to adequate microbial
cell adhesion on the electrode, their experimental findings demon-
strated that the MFC operated with CP/PANI modified with ethyl-
enediamine outperformed the others [121]. Some of the electrode
modifications are tabulated in Table 5.
Together, the inoculum source, reactor configuration, electrode
materials, and operational circumstances all impact on MFC's per-
formance and output. Due to the broad and abundant microbial
populations and their interactions in the EAB, mixed consortia
have become widespread. However, unexplained and frequent
changes in microbial populations throughout an experiment and
adjustments to the media’s nutritional balance affect the systems’
ability to work consistently. Numerous electroactive microbial com-
munities used for bioelectricity production and other potential
uses in BESs have been isolated over time from enriched MFCs.
Some of these electrode materials which could be used and their
pros and cons are discussed below.
6.1. Electrodes Composed of Various Metals
According to various literature on electrode materials, copper, gold,
and silver had the best performance among some metals tested
with variable current densities. Biofilms with polarization potentials
of 0.2 V were developed on silver, gold, and carbon anode materials.
On the other hand, copper, steel, nickel, and titanium anode materi-
als may be developed with a -0.2 V potential. Gold, silver, copper
electrodes, nickel, stainless steel, Titanium, and cobalt, have all
been extensively researched to construct MFCs [122]. Because of
compatibility, silver, copper, and gold provide better current in-
tensities than other metals. Numerous metallic electrodes have
a current transmission magnitude nearly twice that of conventional
graphite electrodes. If economic feasibility is considered, stainless
steel is an enticing resource for MFC as an electrode source due
to its reasonable price and outstanding conductivity. However,
due to the poor biocompatibility of metal electrodes to micro-
organisms, the use of metal electrodes in MFCs is limited; therefore,
thermal oxygenation was used to improve their functional qualities.
6.2. Electrodes Composed of Carbon or Graphite
Over the years, various carbon materials have been utilized in
MFCs, including carbon felt, graphite, carbon cloth, charcoal foam,
graphene, carbon paper, carbon brush, and graphene felt [123].
These materials allow microbes to mediate high-speed electron
transfer, making them an intriguing category of electrode materials
11
Tahseena Naaz et al.
for MFC. In this regard, biofilm and interfacial colonization sig-
nificantly increase when such carbon compounds are utilized, re-
sulting in enhanced power output [124]. Electrochemical pre-proc-
essing with graphene sheets to improve biofilm development has
been mentioned earlier in the literature. Modification of graphite
surface yields carboxyl-containing side chains, which boosts micro-
bial adherence solely at the active electrode material junction and
improve electrochemical characteristics of the MFC. The graphene
oxide layer typically contains atmospheric O2, which forms various
reactive compounds on the graphene sheets layer, including phe-
nolic, quinoline, carboxylic acids, and acetic acids, which promote
charge transfer across the biofilm [125].
6.3. Electrodes Composed of Porous Ceramic
When graphite fiber electrodes were utilized, no biofilms were
generated in some microorganisms, such as Chlorella vulgaris. But
using porous ceramic electrode materials in Chlorella vulgaris, such
as tin oxide with fluorine-doped coating on nanoporous TiO2 ce-
ramic materials, can be employed for MFC biofilm formation. The
fluorine doping TiO2-coated ceramic electrode had a fibrous exterior
network, allowing Chlorella vulgaris to cling to it [126]. As a result,
the EPS became less fibrous. In addition, studies regarding biofilms
produced on carbon felt wires, exposed disoriented cells, and a
lacking EPS are required for biofilms' formation and their growth.
Inside the carbon felt, a dense aggregate of cells of Chlorella vulgaris
was present, but they did not adhere properly to the electrode
[44].
6.4. Electrodes Composed of Titanium
According to the latest studies, the performance of the titanium
(Ti) anode based on the types of materials can be classified as
Ti without any coating > Ti with Pt-coating > or flat graphite
[127]. Even though Ti has great resistance against corrosion, the
poor conductivity of Ti, its scalability, and its less biocompatibility
make it inappropriate for application as electrode material in MFC.
Because of its broad bandgap, TiO2 in nanotubes is widely used
on the positive electrode surfaces of MFC to enhance biofilm
formation. For example, carbon cloth, paint, and graphite felt are
not feasible for photosynthetic biofilm formation. At the same time,
this has been successfully proven by utilizing carbonic barriers
and carbonic sheets [128].
One of the drawbacks of MFCs is that power generation does
not exponentially rise with adequate contact coverage of the electro-
des to accommodate for increased EET. Approaches including low-
ering oxidation and reducing overpotentials, enhancing fluid con-
ductance, decreasing mass transfer resistances, shortening the dis-
tance between electrodes, employing novel gas electrode materials,
and layering have all been used to help scale up the MFCs from
the laboratory level to the industrial level [129]. While improve-
ments in the scaling up of MFCs and their architecture have resulted
in increased power output of the MFC, they have not addressed
the current state of electrode development used in the fundamental
scaling of such devices. The EET techniques employed by EABs
are linked closely to the scaling up of anodes in MFCs and other
bioelectrochemical systems [130]. Considering each electrode’s con-
12
tribution to the overall energy transmission is crucial.
7. Processes Concerning Biofilm Attachment
and Detachment: Challenges
Advective flow and three forms of non-covalent interactions influ-
ence the initial adhesion of microorganisms to a solid surface:
(1) Lewis’s acid-base (L-AB), (2) Lifshitz version of Vander Waals
(LVW) (also called electrodynamic connections), (3) double elec-
trical layer (EL). Such relationships are thought to be in the extended
DLVO theory (EDLVO, where DLVO stands for Derjaguin-Landau-
Verwey-Overbeek) [131]. All liberated energies for each type of
relationship as a distance variable can be calculated and kept sepa-
rate before combining all three (expressed in the same energy forms)
to get the ultimate XDLVO graph [132]. Metal ions with valency
two (Mg2+ and Ca2+) can create ionic bridges between negative
molecules or substances, which could also affect initial attachment.
Cell mobility by flagella might propel cells in routes towards poten-
tial gradient (chemotactic way), electrostatic interactions (electro-
galvanotaxis), electromagnetic field (magneto- taxis), and illumina-
tion (photo-taxis), which also acts as additional forces affecting
initial adhesion [133], when a pioneer species of microbes connects
to a substrate surface, the number of active sites for microbial
adhesion of other species increases.
Several factors influence cell adhesion and biofilm production,
such as roughness, hardness, wettability, cell surface features, and
types of substratum [134]. Flagella, fimbriae, pili, slime layer, capsu-
le production (EPS), as well as the physiochemical parameters
such as nutrients, pH, temperature, O2, etc., consist of cell surface
features. Some of the distinct mechanisms in a biofilm detachment
process are - a) Erosion and shearing, where tiny blotchy sections
of such biofilm are continually eliminated; b) sloughing (rapid
and comprehensive clearance); c) abrasion (detachment owing to
the impact of mass fluid molecules on the biofilm interface); d)
natural shedding. These dispersal models may have an impact
on the species' phenotypic traits. Eroded or sloughed biofilm ag-
gregates are particularly prevalent in denser biofilms that form
in nutritionally rich environments [135]. Several biofilm features,
such as EPS and antibacterial resistance, are likely to be retained
in the biofilm, while colonies shedding as a result of growth may
swiftly convert to planktonic morphology. As expected, the biofilm
detachment rate increases with the depth of the biofilm cover,
associated with severe fluid shearing at the microbial biofilm liquid
interface. Sloughing in this state is much less predictable than
degradation and is thought to be triggered by a lack of nourishment
or oxygen inside the biofilm matrix. At high hydrodynamic shear
stress, harsh granules of minerals in the intake medium may damage
biofilms by abrasion [136].
8. Insertion of Synthetic Amino Acids (AAs) to
Improve the Performance
Within the Schultz laboratory [137], tools for integrating artificial

amino acids (AAA) onto proteins of bacteria in vivo have been
developed. By employing tRNA-NH2-acyl tRNA synthetase pair-
ings, this approach allows for the preferential integration of unusual
amino acids (AAs) into polypeptides in yeast [138], mammalian
cells [139], and E. coli [140]. These pairings do not bridge with
intrinsic parts of translating mechanism but rather identify the
target AAA and integrate this into proteins with reaction to TAG,
a nonsense codon in yeast and E. coli, or TGA, another opal codon
in animal cells. To improve system selectivity, tRNA synthetases
are now being created to integrate only AAAs into proteins while
ignoring native AAs. Interaction between redox enzymes and the
electrodes can be increased by adding different AAAs upon that
site of the enzyme, allowing electrons to transit between the enzy-
me's active site and its surface and then to the electrodes. Redox
enzymes would gain stabilization and stay functioning for prolonged
periods if they are expressed at the surface, which offers a stabilizing
microenvironment for the microbes. 3-amino tyrosine has been
recently integrated further into E. coli RR (ribonucleotide reduc-
tase)'s A2 subunit, and it has been conclusively proved that 3-AT
(aminotyrosine) continues to serve as a radical pit instead of a
single tyrosine radical, showing the presumed electron transfer
route in this enzyme involved [141]. This study has also provided
evidence for immediate electron transport communication between
the redox AAA and the sulfur-iron group in this enzyme, which
motivates us to use AAA as electron transport.
9. Bacteria Coated with Gold and Silver
Nanoparticles
Studies have utilized nanoparticles (NPs) as electrical bridgers be-
tween the electrodes and enzymes' active sites [142]. The Willner
group achieved an eight-fold increase in electron turnover rate
from the original enzyme's cofactor to its natural receiver. FAD-re-
formed GOx was employed, where the FAD got altered with gold
nanocrystals and subsequently connected to electrodes. Its align-
ment concerning the electrode aided in achieving the highest trans-
fer of electrons possible. Over the years, various approaches have
been used to proficiently wire oxidation/reduction enzymes, such
as encapsulation in conjugated polymers [143], trying to combine
nanotubes with redox enzymes [144,145] or even site-targeted muta-
genesis to ensure alignment of proteins with diodes146. Several
attempts to cover microorganisms with metallic NPs have been
attempted. One example is the manufacture of gold nanoparticles
(GNPs) with the help of E. coli, in which the use of microbiological
NP hybrids was proven in the direct electrochemical processes
of hemoglobin [147]. There is a distinction between the manufacture
of GNPs by microbes and the adhesion of GNPs generated in an
inorganic setting and afterward attached to the bacteria. Tufted
organisms with two lengths of fibrils, such as Streptococcus sanguis,
contain a charge and thus are hydrophobic on their short and
long filament fibrils [148]. So, because the bald region of the surface
of the microbes is hydrophilic, colloid gold, which is hydrophobic
and is attracted to the negative, did not cling to it throughout
a pH of 3.7 to 9.0. The attachment of colloidal gold to lengthy
13
Environmental Engineering Research 28(5) 220666
fibrils happened regardless of pH. When the pH is 3.7, colloidal
gold adhered to short fibrils. The electrostatic interaction between
the negative charges short fibrils and GNPs grew at higher pH
levels, preventing colloidal gold from attaching to the short fibers
[149]. In the mid-1980s, it was discovered that bacteria collect
metals like gold, and silver, which are recoverable from various
bacterial species by methods that are yet unknown [150]. Many
research groups have recently proven the production of GNPs by
various bacterial species [151–153]. The Raman spectra of silver
metal produced by bacteria were reported by Zeiri et al. The bacteria
were exposed to sodium borohydride, which acted as a nucleating
substrate for silver ion reduction, and a rough silver coating devel-
oped all around microorganisms [154]. Several microorganisms
have been employed to generate defined as follows nanostructures
composed of metallic nanoparticles [149,155,156]. R. F. Saraf's team
applied extremely negative charges of the teichoic acid brush to
the surfaces of the Gram -ve bacterium Bacillus cereus.
Furthermore, nanorod-coated bacteria have shown greater con-
ductivity than nanospheres at just approximately 10% coverage,
far below the threshold of almost 45%. The high electrical con-
ductivity makes it possible to build electronic circuitry on top
of the bacterium without smothering it [156]. These findings imply
that site-specific adhesion of nanorods to microorganisms may result
in more efficient electron transport between bacteria and electrodes
than spherical attachment would.
10. Advances in Biofilm Engineering to Enhance
the MFC Performance
Given the importance of EPS in microbial EET and biofilm develop-
ment on the electrode, controlling the biofilm formation techniques
for enhanced adherence and EET is indeed the MFC's future goal.
Several MR-1 excision mutants of Shewanella oneidensis were gen-
erated and evaluated for electricity production and oxide reduction
[157]. Shewanella oneidensis MR-1’s EET process includes MtrC,
OmcA, and decaheme ccyts in transferring electrons to heavy steel
oxides and MFC electrode materials; however, Shewanella loihica
PV-4 exhibited a distinct method for current production [158].
In another study using the Shewanella oneidensis MR-1 strain,
it was found that the conducting EPS found in the biofilm affects
the cell attachment to the graphite anode material and current
production in MFCs. At the same time, the electromagnetically
non-conductive polysaccharides can start interfering with the elec-
tron transfer when in contact with cytochromes. Cell surface treat-
ment has therefore been offered as a potential technique for boosting
power in an MFC system [159]. In a model organism Shewanella
oneidensis MR-1, genetic engineering approaches were used for
the biosynthesis of flavin metabolizing enzymes gene cluster ribC-
ribD -ribE-ribA and a metal-reducing channel biosynthetic gene
group mtrA-mtrC-mtrB, resulting in an increased EET rate in MFC
with approximately 110% increase in the current production [160].
Similarly, the use of partial degradation of graphite composite
substrate by Ultraviolet/Ozone exposure resulted in changes in
the surface properties of the electrode, allowing for enhanced EAB
Tahseena Naaz et al.
growth on the electron, which increased EET. This Lead to high
power production in MFC. Shewanella oneidensis MR-1 microbial
community establishment was advanced on UV/O3-medicated
graphite felt anode and cathode at 0.3V versus Ag/AgCl, with the
graphite terminals of electrodes treated to 45 minutes of ultraviolet
and ozone intervention which improved the microbial adhesion
on the electrode surface [161]. Furthermore, the influence of specific
working conditions on biofilm development and nitrogen fixation
in three moving-bed biofilm reactors (MBBRs) was also investigated.
Scientists used genetics and gene suppression techniques to study
the relevance of such attachments, revealing a comprehensive de-
piction of the EET pathway from bacteria to the positive electrode.
Yet, minimal reports of phytoplankton that were genetically modi-
fied or biochemical processes of phytoplankton, along with their
voltage productivity in energy generation, were reported.
However, enhancing EPS synthesis using modification in the
substrate strategies has boosted power output in phytoplankton
[30]. Aside from its role in the complex creation of amylopectin
and the development of starch synthase, new capsules, a GT5 en-
zyme, have been studied because of their contribution to carbohy-
drate synthesis. There is currently no indication that these poly-
mer-producing enzymes are being upregulated in algae at the
moment. Transcription factors responsible for EPS production in
microalgae would thus require extensive investigation before be-
coming a feasible strategy for enhanced power production in photo-
synthesis processing algae MFC (PAMFCs), which are more lasting
than MFC.
The science of bio-electrochemistry has faced a significant issue
for many years: improving the current generation of MFCs. There
have been numerous papers and patents as a result of this endeavor.
Electrode materials, electrode spatial layout, electrolytes, current
collectors, reactor architecture, separation membranes, and micro-
bial catalysts are just a few of the MFC components that have
been optimized. The three methods, screening, adaptation, and
synthetic biology, are frequently utilized to create effective micro-
bial catalysts for MFCs. Four categories can be made from the
synthetic biology techniques used to increase the EET rates between
microorganisms and anodes:
• optimization of substrate transport and cytoplasmic metabo-
lism of electrogenic microbes,
• reengineering of DET mechanisms,
• reengineering of IDET mechanisms,
• optimization of cell adhesion and biofilm formation
To increase the availability of electrons in the form of NADH
for subsequent EET to electrodes, the first technique manipulates
the electrogenic microorganisms' core metabolism. DET and IDET
in the second and third techniques can be strengthened and opti-
mized to increase the flux of electrons from the cytoplasmic mem-
brane to the electrode. Finally, strong micrometer-thick biofilms
can be formed by electrogenic bacteria such as Geobacter spp.
[162]. EET proteins are used by cells from the biofilm's edge to
the electrode surface to generate current. Therefore, microorganisms
that have been altered to be better at attaching to cells and forming
biofilms ought to be better at transferring electrons to the anode.
Microbial adherence to the anode surface is critical in boosting
MFC’s productivity and higher energy production [163].
14
Pseudomonas aeruginosa’s biofilm formation and cell attachment
ability improved when genetic manipulation was done. PilT mutants
in P. aeruginosa lack twitching motility because their pili are perma-
nently stretched [164]. PilT is an ATPase required for the depolyme-
rization of pilin subunits in P. aeruginosa type IV pili, which are
non-conductive [165]. Type IV pili concentrate on the surface of
less mobile P. aeruginosa cells in the absence of PilT, resulting
in increased biofilm development, a cell with cell adherence, and
cell adhesion to electrodes [164].
To date, no reports of phytoplankton that have been genetically
altered or that have biochemical pathways, as well as their potential
productivity in energy cultivation, have been reported. However,
improving EPS synthesis utilizing substrate modification ap-
proaches has been demonstrated to enhance power production
in phytoplankton. Starch synthase, a glycosyltransferase 5 (GT5)
enzyme, has been looked at for its potential role in increasing
carbohydrate production and its involvement in the complex for-
mation of amylopectin and the creation of new capsules. There
is yet no proof that these polysaccharide-producing enzymes in
algae are being up-regulated. To date, no reports of phytoplankton
that have been genetically altered or that have biochemical path-
ways, as well as their potential productivity in energy cultivation,
have been reported. However, improving EPS synthesis in an in-
dividual utilizing substrate modification approaches has been dem-
onstrated to enhance power production in phytoplankton. Starch
synthase, a glycosyltransferase 5 (GT5) enzyme, has been looked
at for its potential role in increasing carbohydrate production and
its involvement in the complex formation of amylopectin and the
creation of new capsules. There is yet no proof that these poly-
saccharide-producing enzymes in algae are being up-regulated.
The attempts to improve the performance of electroactive bacteria
have focused on altering the genetics of the EET phenomenon
due to developments in genetic engineering and synthetic biology.
The rate of electron transfer and the efficiency of electroactive
bacteria is generally low, except for Shewanella and Geobacter.
Due to the rapid development of analytical tools and advancements
in biotechnology, researchers have started changing strains to create
modified electroactive bacteria to improve electron transport.
Exoelectrogens that have been developed have also been found
to improve target product performance and achieve various bio-
process goals in microbial system-based processes. The power den-
sity is doubled when glycerol dehydrogenase (GldhA) is overex-
pressed in genetically altered Escherichia I compared to wild-type
ones. This was due to the production of redox mediators from
the GldhA-catalyzed metabolism of aminopropanol, threonine,
L-1,2-propanediol, and glycerol [54]. In similar research, heteroge-
neous overexpression of cyclic-di-GMP in Shewanella sp. dramati-
cally increased biofilm formation, resulting in almost threefold
greater power density output 166. A non-metal-reducing E. coli
strain created by adding MtrCAB genes from Shewanella oneidensis
can reduce metallic ions eight times greater than the original strain
conduit [167]. The expression of key enzymes for glucose and
glycerol fermentation and metabolite synthesis is influenced by
electrode-based electron transfer in MFCs [168]. In producing effec-
tive electroactive bacteria, a recent study of engineered electrogens
emphasized the techniques, genetic modification of electrochemical
 Environmental Engineering Research 28(5) 220666

Fig 2. Electron transfer (EET) and electronic regulation in non-native hosts engineered via genetic engineering. For an orthogonal expression to
occur in a non-native host, specific conditions must be met using engineered expression vector transfer (EET) pathways: i) Maintaining
correct cytochrome expression levels. ii) The correct cytochrome c maturation process allows optimal protein expression and localization
without causing unnecessary toxicity. iii) Proper folding of the proteins that were expressed into their complexes.

genes, and a synthetic biology approach [169].
11. Strategies of Synthetic Biology
Microbial catalysts, separation membranes, reactor configuration,
current collectors, electrolytes, electrode spatial design, and elec-
trode materials have all been optimized in MFCs to enhance their
performance. Four categories can be established for the synthetic
biology approaches used to increase ET rates between the anode
and the microbes [170]:
• Electrogenic bacteria’ cytoplasmic metabolism and substrate
transport are optimized.
• Improvements to the Direct EET Mechanism.
• Improvements to the indirect EET Mechanism (Fig. 3).
• Development of biofilm formation and cell adhesion.
The first case involves modifying the core metabolism of electro-
genic bacteria to increase the amount of NADH available for EET
to the electrodes. The EET mechanisms can be optimized in the
second and third approaches to increase the flow of electrons to
the electrode from the cytoplasmic membrane [171,172]. Last but
not least, exoelectrogens like Geobacter spp. can produce biofilm
of several micrometers in thickness. Cells from the biofilm's periph-
ery to the electrode surface can produce current using a system
of extracellular ET proteins. Thus, microorganisms engineered to
bind to cells and produce biofilms more effectively should be better
at electron transfer to the anode. Antibiotic selection markers, plas-
mid shuttle machinery, Promoters, and replication origins have
been recently identified in G. sulfurreducens and S. oneidensis
and can be employed in either the orthogonal or native host to
regulate EET pathways [173]. The severe anaerobic cultivation re-
quirements of G. sulfurreducens have hampered the speed of bio-
engineering, despite the availability of a molecular toolbox for
G. sulfurreducens. To modify the genome of S. oneidensis with
great precision and specificity, using ssDNA oligos was necessary,
and creating a highly efficient electrotransformation process made
this possible. Corts et al., who attempted and optimized recombin-
eering technology in S. oneidensis, found that ssDNA oligos can
recombine site-specifically in the chromosome of S. oneidensis
in the control reactions in the absence of recombinase but at a
somewhat lower frequency than W3Beta-mediated recombination
[173]. CRISPR/Cas9 is another step forward in genome engineering
for S. oneidensis. In order to get rid of the unedited cells once
recombineering was produced, Corts et al. employed CRISPR/Cas9
[174]. By targeting a specific wild-type (unedited) gene in the chro-
mosome with a single guide RNA, Cas9 may create a double-stranded
break that is lethal for many bacteria, making it a powerful tool
for use in counter-selection [175].
The expression of numerous multiheme cytochromes c was a
significant technological barrier in developing EET-capable E. coli
strains (Fig. 2). Kinetic equilibrium between maturation, secretion,
translation, and transcription is necessary for multiheme c cyto-

15
Tahseena Naaz et al.

Fig 3. The efficiency of microbial fuel cells (MFCs) can be enhanced by using synthetic biology to target specific cellular processes of microorganisms.
(a) Increased substrate uptake is achieved by overexpressing the transporter protein. (b) Substrate oxidation speedup by genetic engineering.
(c) Genetic modification of central metabolism to boost NADH/NAD+ ratio or pool. (d) Regulatory network's genetic modification or phenazines’
biosynthesis-related gene overexpression to boost the production of phenazines. (e) Soluble electron shuttle (ES) transport via the cell wall
can be improved by overexpressing porin. (f) Improving direct extracellular electron transport by c-type cytochrome’s overexpression. (g)
Redox enzyme's surface display. (h) The synthetic biology method improves biofilm growth and cell adhesion to an electrode surface.

chromes expression [176]. CcmH recognizes and binds the apo
cytochrome c protein, which then undergoes a thioether bond for-
mation with heme b to create the holoprotein [177], which is the
end product of the cytochrome c maturation (ccm) system.
12. Research Directions for Electroactive
Biofilms in the Future
A first step could be to look into different measuring methods
for quantifying electron fluxes and archiving in EAB.
Electrochemical tests should preferably be carried out in combina-
tion with visual observations obtained in the field to measure the
surface area and biofilms' densities as a variable dependent on
time [178]. This would enable the study of bioactive compounds
per unit of organic matter under various situations. CLSM (confocal
laser scanning microscopy) [179] and Raman spectroscopy [180]
are two techniques that could be used to determine which parts
of the cell are essential for electron storage and transit.
Across these lines, UV-Vis light spectroscopy [181] and fluo-
rescence spectroscopy could be used to evaluate the cytochrome
heme concentration and the degree of cytochrome pool declines.
Hence, the redox state of cytochromes can be used for predicting
energy output [182]. Likewise, cyclic voltammetry may calculate
the mediatory voltage of operational charge carrier cell components
[183]. Furthermore, combining such methodologies allows the prop-
erties of various components of MFCs to be studied in greater
depth [184].
Accessibility of the electrolytes and electrogenic bacteria in MFCs
is a fresh field of investigation with many possibilities [185]. In
this aspect, oxygen has the potential to break down complex organic
material into power-generating compounds as by-products. It is
also critical to assess the feasibility of mutualistic relationships
between stringent anaerobic organisms and aerotolerant anaerobic
microorganisms and the biofilm role in safeguarding electrodes
against contamination [116]. One example can be researching the
EET processes in somewhat under aerobic conditions. These themes
should be the focus of exciting research in the future. Another
set of concerns emerges from using MFC technology to generate
power and by-products by treating wastewater as a commodity
while also seeking to collect and treat wastewater [186–190].
EABs’ ability to oxidize a wide range of biomolecules and the
transport of electrons are some critical aspects. Furthermore, con-
verting specific and complex natural substances are significant
issues that may demand pretreatment to be used as an anolyte
in MFC to produce power [124]. Research in this field will increase
as systems achieve commercially relevant performance. Although
much is known about the methods allowing EABs to transfer elec-
trons toward the positive electrode, there are numerous
uncertainties. The significance of biofilm structure and its compo-
nents, including EPS and nanotubes, in regulating electron con-
ductivity in EAB is unknown; nonetheless, knowing how they oper-
ate and how they could be improved is crucial. The utilization

16

of fluid chambers based on microplates and miniature MFCs, in
combination with current- and voltage-sensitive pigments and com-
plex bio-molecules, appears to possess the capacity to simplify
research, offering a better knowledge of EABs’ role in MFC. Research
regarding biocathodes, whether the microorganisms at the cathode
are electro-chemolithotrophs or mixotrophs, is underway [191].
13. Conclusion
This paper describes various ways to improve biofilm formation
to increase the effectiveness of MFCs and generate high current
output. Under this situation, biofilm engineering may enhance
electron transport in electrode-electrolyte conjunctions. Improved
microbial activity and electrical conductivity can lower charge
transmission resistance at the anode. Furthermore, new materials
for electrolyte and electrode functionalization technologies could
significantly help in customizing biofilms and increasing rates of
bio-electrocatalysis. Exogenous supplementation of biological me-
diators that boost the signaling cascades necessary for biofilm estab-
lishment can improve biofilm development. The final strategy,
which involves manipulating genes encoding biofilm-related pro-
teins, necessitates expertise and elegance. An intelligent blend of
the physicochemical, biological, and computational approaches
discussed in this review will need to be applied to improve biofilm
growth and activity for specific applications. However, significant
obstacles remain until electrogenic microorganisms for MFCs may
be significantly genetically engineered in a custom-tailored way.
However, our understanding of the EET mechanism of micro-
organisms to anodes remains inadequate, despite tremendous ad-
vances in this area in recent years. It was also discovered that
MFCs might be used to reliably power low-power medical equip-
ment implanted in humans over the long term. These miniaturized
MFCs, bio-catalyzed by Saccharomyces cerevisiae, can convert the
glucose from the blood into electricity. It was discovered that the
current generation rises in synchrony with the level of pollutants.
Consequently, this method can help detect and count microbes
in food [192]. It is necessary to thoroughly understand the metabolic
activity and anatomy of electrogenic microorganisms to develop
effective synthetic biology techniques. Nonetheless, the work done
so far is impressive, and it appears that using synthetic biology
methods gives us a chance to enhance the performance of the
MFC as well as genuinely comprehend it.
Author Contributions
Conceptualization. S.P.J, and S.P.; writing—original draft prepara-
tion, C.P, A.K, T.N, and S.P.J; writing—review and editing, N.S,
K.P and S.P.; supervision, S.P, K.P and S.P.J; funding acquisition,
S.P.J. All authors have read and agreed to the published version
of the manuscript.
Acknowledgements
This work was supported by the National Research Foundation
17
Environmental Engineering Research 28(5) 220666
of Korea (NRF) grant funded by the Korean government (MSIT)
(NRF-2021R1A2C1013989).
Conflicts of Interest
The authors declare no conflict of interest.
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