Bioengineered

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/kbie20

Sustainable circular biorefinery approach for novel

building blocks and bioenergy production from
algae using microbial fuel cell

Kevin Tian Xiang Tong, Inn Shi Tan, Henry Chee Yew Foo, Pau Loke Show,
Man Kee Lam & Mee Kee Wong

To cite this article: Kevin Tian Xiang Tong, Inn Shi Tan, Henry Chee Yew Foo, Pau Loke Show,
Man Kee Lam & Mee Kee Wong (2023) Sustainable circular biorefinery approach for novel
building blocks and bioenergy production from algae using microbial fuel cell, Bioengineered,
14:1, 246-289, DOI: 10.1080/21655979.2023.2236842

To link to this article: https://doi.org/10.1080/21655979.2023.2236842

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and managed by Curtin (Malaysia) Sdn Bhd
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BIOENGINEERED
2023, VOL. 14, NO. 1, 246–289
https://doi.org/10.1080/21655979.2023.2236842

Sustainable circular biorefinery approach for novel building blocks and

bioenergy production from algae using microbial fuel cell
a a a b,c,d,e f,g
Kevin Tian Xiang Tong , Inn Shi Tan , Henry Chee Yew Foo , Pau Loke Show , Man Kee Lam ,
and Mee Kee Wong h
a
Department of Chemical and Energy Engineering, Faculty of Engineering and Science, Curtin University Malaysia, Miri, Sarawak, Malaysia;
b
Department of Chemical Engineering, Khalifa University, Abu Dhabi, United Arab Emirates; cZhejiang Provincial Key Laboratory for
Subtropical Water Environment and Marine Biological Resources Protection, Wenzhou University, Wenzhou, China; dDepartment of Chemical
and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Malaysia; eDepartment of
Sustainable Engineering, Saveetha School of Engineering, SIMATS, Chennai, India; fChemical Engineering Department, Universiti Teknologi
PETRONAS, Seri Iskandar, Perak, Malaysia; gHICoE-Centre for Biofuel and Biochemical Research, Institute of Self-Sustainable Building,
Universiti Teknologi PETRONAS, Seri Iskandar, Perak, Malaysia; hPETRONAS Research Sdn Bhd, Kajang, Selangor, Malaysia

ABSTRACT ARTICLE HISTORY

The imminent need for transition to a circular biorefinery using microbial fuel cells (MFC), based Received 24 April 2023
on the valorization of renewable resources, will ameliorate the carbon footprint induced by Revised 23 June 2023
industrialization. MFC catalyzed by bioelectrochemical process drew significant attention initially Accepted 11 July 2023
for its exceptional potential for integrated production of biochemicals and bioenergy. KEYWORDS
Nonetheless, the associated costly bioproduct production and slow microbial kinetics have con­ L-lactic acid; microfluidic;
strained its commercialization. This review encompasses the potential and development of electrofermentation;
macroalgal biomass as a substrate in the MFC system for L-lactic acid (L-LA) and bioelectricity bioelectricity; 3D printing
generation. Besides, an insight into the state-of-the-art technological advancement in the MFC
system is also deliberated in detail. Investigations in recent years have shown that MFC developed
with different anolyte enhances power density from several µW/m2 up to 8160 mW/m2. Further,
this review provides a plausible picture of macroalgal-based L-LA and bioelectricity circular
biorefinery in the MFC system for future research directions.

CONTACT Inn Shi Tan tan.s@curtin.edu.my Department of Chemical and Energy Engineering, Faculty of Engineering and Science, Curtin University
Malaysia, Miri, Sarawak CDT 250, 98009, Malaysia; Pau Loke Show PauLoke.Show@ku.ac.ae Department of Chemical Engineering, Khalifa University,
Shakhbout Bin Sultan St - Zone 1, Abu Dhabi, United Arab Emirates
© 2023 Curtin University, Malaysia Is owned and managed by Curtin (Malaysia) Sdn Bhd 199801008086 (464213-M). Published by Informa UK Limited, trading as Taylor & Francis
Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the
Accepted Manuscript in a repository by the author(s) or with their consent.

1. Introduction
Industrialization, the engine of urbanization and
economic expansion, has accelerated the develop­
ment of petrolic energy and polymer sectors in
association with the growth of the global popula­
tion and affluence [1]. Meanwhile, due to the
outbreak of the COVID-19 pandemic, a nearly
130-fold increment of plastic waste has been per­
ceived over the past 10 years (2011–2020) and
reached approximately 8.51 billion tons as an
unprecedented increase in single-use plastics
(SUPs), including protective medical suits and
face masks [2]. It is assessed that mismanaged
plastic wastes are omnipresent and outweigh fish
in the marine environment, with at least 7 million
tons of plastic debris dumped into the ocean
annually, which has caused catastrophic damage
and deaths of marine animals [3]. Such vivacious
SUPs production results in well-established con­
cerns that are deleterious to the environment,
such as land pollution, air pollution, and marine
pollution that causes anthropogenic CO2 emis­
sions. The International Energy Agency (IEA)
website shows that the rise in carbon footprint
from 2005 to 2021 has reached an 18% increment
[1]. This linear economy has been heavily reliant
on the overexploitation of fossil fuels and natural
resources, which drastically impairs the life of
future generations [3]. Suppose the carbon foot­
print continuously rises at its current rate into the
atmosphere. In that case, the dynamic equili­
brium of the carbon cycle will diverge, resulting
in irreversible changes in the climate system.
Hence, sustainable practices and concerted efforts
to minimize carbon footprint whilst simulta­
neously maintaining global economic growth
have to be initiated through various technological
interventions to meliorate the current linear
economy toward a biobased bioeconomy.
In pursuing the United Nations’ 7th Sustainable
Development Goal (SDG), the bioeconomy has to
be designed to generate affordable, clean, and sus­
tainable energy [4]. From the point of view of
sustainability, this concept covers three major
points: (1) maximum utilization of organic
resources; (2) minimum energy suffices the pro­
duction processes; (3) the waste produced from
BIOENGINEERED 247
one operation becomes the feedstock for another
[5]. Notably, the circular bioeconomy model has
drawn significant attention to address the environ­
mental burden and resource depletion caused by
the linear economy model. In the circular bioec­
onomy model, the maximum energy potential and
value of the biomass can be aroused, extracted,
and retained to their maximum extent via the
circular biorefinery approach. The circular biore­
finery approach is a regenerative closed-loop
method that conserves the longevous biomass
resources to maximize economic productivity and
minimize waste generation at the end of each
service life, which helps mitigate the pressure on
the environment [6]. Recently, the concept of hol­
istic zero-waste circular biorefinery has been suc­
cessfully applied to the co-production of
bioethanol and biogas (biomethane) from red
macroalgae Eucheuma denticulatum residues by
Loh et al. [7]. They concluded that the developed
macroalgae-based circular biorefinery system
demonstrated an exergy efficiency of 73.74% with
a functional exergy efficiency of 15.30% and the
cascading approach allowing an almost complete
conversion of the macroalgal biomass [7].
A macroalgae-based circular biorefinery system
for bioethanol and L-lactic acid (L-LA) was also
established by Wong et al. [8] using red macro­
algae Eucheuma cottonii residues, manifesting the
electricity generated from the combined heat and
power plant of the system proficient at supplying
up to 70% of the plant’s total electricity require­
ment. Further, Grasa et al. [9] investigated the
economic evaluation using brown macroalgae
Laminaria sp. as biomass for large-scale L-LA
production. They revealed that the minimum
viable selling price is reduced by 34% compared
with the current selling price (USD 3.83/kg),
demonstrating an economic feasibility and sustain­
ability biorefinery approach [9]. To this extent, the
macroalgae biomasses tally with the circular bior­
efinery concept compared to lignocellulosic bio­
mass (LCB), a cornerstone of the biorefinery to
date due to non-competitiveness with food supply
and fast growth rate [10,11].
To achieve carbon neutrality and a sustainable
circular biorefinery, durable biobased products
need to be developed without sacrificing
248 K. T. X. TONG ET AL.
performance. Various strategic paths to produce
carbon-neutral macroalgal bioenergy and bioplas­
tics have been mapped out with expanding
research outputs [12,13]. At the upshot of these
efforts, one of the latterly proposed alternative
bioenergy sources is microbial fuel cells (MFC)
which generate renewable electricity using active
microorganisms or bacteria as a biocatalyst in an
anaerobic anode chamber [13]. Gebreslassie et al.
[13] revealed that brown macroalgae Saccharina
japonica was successfully metabolized by metha­
nogenesis in a H-shape dual-chamber MFC for
bioelectricity production with a maximum power
density of 1820 mW/m2. Further, Okoroafor et al.
[14] detailly calculated the substitution potential of
petrolic electricity with bioelectricity produced via
MFC and evaluated global warming potential
(GWP) impacts. Initially, the GWP was evaluated
based on the United Kingdom’s electricity
demand; further, GWP for bioelectricity MFC
was evaluated for petrol-based electricity. It has
been deduced from the calculations that 15.7–
19.5 ktons of CO2 equivalents can be diminished
by bioelectricity production of 10,030 GJ each year
[14]. Furthermore, using renewable carbon in
bioelectricity helps curtail the dependency on fos­
sil fuels, which can further control or reduce the
carbon footprint throughout this life cycle [15].
In addition to tremendous efforts to reverse
global climate change, a valuable novel building
block for poly-L-lactic acid (PLLA) bioplastic,
notable L-LA, can also produce from macroalgal
biomass via the biotechnological approach by
using lactic acid bacteria (LAB) to valorize carbo­
hydrate contents [11]. PLLA is a biodegradable
thermoplastic with properties similar to petrolic
plastics (polypropylene, polyethylene, polystyrene,
and polyamide) and offers added advantages due
to generating a lesser carbon footprint on the
environment with a feasible end-of-life strategy
[16]. By the biorefinery principle Task 42, the co-
production of bioenergy and value-added bio­
chemicals from biomass in an integrated system
are advocated to advance the material wealth of
human society in terms of materials and energy
supply for a new sustainable industrial develop­
ment [17]. In an evaluation of the carbon footprint
of biorefinery producing biochemicals and bioe­
nergy from wastewater in an MFC model using an
attributional life cycle assessment (LCA) assay, the
authors revealed that the best scenario was the
integration of the production chain compared to
standalone production, resulting in a 112.18%
diminution to the net system emission by attaining
−2.2 global warming ratio compared to standalone
processing [18]. Thus, a cascading macroalgal
biorefinery system in the MFC model is recom­
mended for a zero-waste conversion technology to
address the dilemma of bioelectricity and L-LA.
Once fully optimized, the cascading macroalgal
biorefinery system has the potential to transform
the energy and polymer sector into a climate-
neutral hub whilst helping the circular
bioeconomy.
This paper is systematically designed to criti­
cally review the successful development of bioelec­
tricity and L-LA from macroalgal biomass for
bioenergy and bioplastic applications. The pro­
spects and state-of-the-art technological develop­
ment of MFC for integrated bioprocesses as
a strategy for carbon neutrality and sustainability
were enclosed. In addition, the advantages of key
bioprocesses that are employed for macroalgae
valorization for L-LA and bioelectricity production
were also discussed extensively. Based on the dif­
ferent technological employment, some recom­
mendations were made for future research
directions on the seamless integration of macro­
algal-based L-LA and bioelectricity production.
Hence, this review provides essential technical
enlightenment on the contemporary status and
future trends of MFC applications in pursuit of
a sustainable circular biorefinery.
2. Marine macroalgae: the future of
sustainable bioenergy and bioplastic
The research interest in utilizing marine and cul­
tivated macroalgal biomass began to arise once
hindrances associated with LCB emerged, mainly
the harsh thermochemical pretreatment involved
in removing the lignin complexes [19]. Among
29,800 identified macroalgae strains, merely 230
strains have been studied for biorefinery, and
their potential for miscellaneous applications is
irrefutable [20]. The cradle-to-gate delineation of
the development of macroalgal bioelectricity and
bioplastics is illustrated in Figure 1. Macroalgae

Figure 1. Schematic representation of macroalgal bioelectricity and bioplastics from cradle-to-gate perspectives for durable
applications.
are aquatic photosynthesis and carbohydrate-rich
multicellular eukaryotic organisms ubiquitously
present in marine habitats, hence the acronym
‘marine plants’ or ‘seaweeds.’ Macroalgae are of
three types based on the natural pigmentation
type: red (Rhodophyta) taxonomical group colored
by phycoerythrin, brown (Phaeophyta) taxonomi­
cal group colored by fucoxanthin, and finally
green (Chlorophyta) taxonomical group colored
by chlorophylls. All taxonomical groups contain
specific cell wall polysaccharides such as cellulose
and phycocolloids in varying compositions, which
contribute to the diversity of macroalgal species.
Floridean starch, laminarin and amylopectin are
usually the storage polysaccharides for red, brown,
and green macroalgae, respectively. Their lack of
recalcitrant lignin complexes indicates that less
energy-intensive bioprocesses can be employed to
recover value-added bioproducts of commercial
interest, favoring techno-economic and LCA ana­
lyses of any potential biorefinery processes that
utilize macroalgae as feedstock [8,10]. Moreover,
high and specialty easy-degradable polysaccharides
(65–95 wt%) in each taxonomical group of macro­
algae species offer extensive features for either
platform compounds or direct use in the biorefin­
ery [21].
From the perspective of bioelectricity genera­
tion and L-LA fermentation, the significant dis­
parity between the three macroalgae species relies
upon their inherent sugar composition, and the
conversion yield will be evaluated by the
BIOENGINEERED 249
fermentation performance of the endowing lactic
acid bacteria on the derived rare sugars. Figure 2
depicts the major macroalgae-derived rare sugars
via hydrolysis, revealing that rare sugars in the
form of D-glucose can be found in all macroalgae
taxonomical groups representing the appearance
of cellulose compounds in the macroalgal cell
wall [11]. Red macroalgae, with Gracilaria sp.
and Eucheuma sp. as the representative species,
possess agarose and carrageenan as the heteroge­
neous phycocolloids composed of agarobiose,
D-galactose, and 3,6-anhydro-D-galactose, of
which 3,6-anhydro-D-galactose is non-metabolize
[12,22]. Brown macroalgae are copiously culti­
vated for the extraction of alginate, laminarin,
mannitol, and fucoidan, which is widespread in
food processing applications. L-fucose,
D-mannose, D-galactose, mannuronic acid, and
glucuronic acid are the major monosaccharides
in brown macroalgae hydrolyzates [23,24]. In
addition, green macroalgae mainly embrace ulvan
as the sole heterogenous polysaccharide composed
of L-iduronic acids and rare sugars, including
L-rhamnose and D-tagatose [25]. Thus, macroal­
gae are considered sustainable sources of rare
sugars for biorefinery purposes. It also addresses
the sustainability concerns related to food security
and arable land suffered by the edible crops and
LCBs, as macroalgae are abundant in supply and
generally grow in marine habitats [11].
Furthermore, macroalgae are considered a year-
round available feedstock for biorefinery purposes
250 K. T. X. TONG ET AL.
Figure 2. Major monosaccharides present in the hydrolysates of red, brown, and green macroalgae.
due to their bioavailability properties with a short
average growth period of 10–15 days [26]. The
global industry exploits 35.08 million tons of 230
macroalgae species cultivated worldwide in 2020,
dominated by red macroalgae species, which
seizes 18.84 million tons and corresponds to
53.70% of the total productivity [27]. Besides, the
global annual production of wild stock macroalgae
in seashore without any farming techniques
bestows a net primary production (NPP) of 1.32
Pg carbon per year, where this productivity is
aligned with the Amazon rainforests. NPP is
defined as the balance between photosynthetic
CO2 sequestration and CO2 released by auto­
trophic respiration, in which the positive NPP
value describes the environment possessing high
initial biomass and higher biomass productivity
[28]. Alongside macroalgae propagation from
aquaculture or wild stocks as the feedstock for
biorefinery purposes, carbon offsetting can occur
simultaneously by photosynthetic CO2 sequestra­
tion. This is mainly due to macroalgal habitats
being the most extensive and productive of all
coastal vegetated ecosystems. Raven [29] revealed
that strongly autotrophic macroalgae communities
globally sequester approximately 1.6 gigatons of
carbon/year through their net production.
Regarding climate change, Seghetta et al. [30]
concluded that the net removal of CO2 from the
atmosphere by macroalgae aquaculture and bior­
efinery is about 190 kg of CO2 equivalents per
hectare of cultivation area. From the perspective
of CO2 sequestration, the intense photosynthetic
activity by macroalgal forests obviates ocean

acidification, resulting in a balanced pH for mar­
ine ecosystems. In fact, ocean acidification repre­
sents a threat that negatively impacts the growth
and survival of calcifying organisms, including
coral reefs, oysters, shrimps, and clams by wrest­
ing the minerals used by these organisms to build
their shells and skeletons [31].
In addition, bioenergy and bioplastics produced
from macroalgal biorefinery will be zero net addi­
tion of CO2 to the atmosphere; any CO2 emitted in
combustion originated from that sequestered dur­
ing recent photosynthesis and can potentially be
recaptured in the same way [1]. Moreover, Plastics
Europe Association (PEA) conducted the LCA of
macroalgal-based bioplastic and concluded that
macroalgae biomass has substantial potential to
produce value-added biochemicals such as L-LA
for bioplastic production [32]. In short, the round-
of-the-year availability of macroalgal biomass, the
ability for CO2 sequestration, and high carbohy­
drate content increase their potential for bioenergy
and bioplastics production.
2.1. Bioenergy
Interest in biomass-to-energy (B2E) strategies has
peaked in the quest for establishing a carbon neu­
tral cycle by drawdown CO2 released, solving both
energy and environmental crises simultaneously.
Figure 3. Microbial fuel cells (MFC) for bioelectricity production from a variety of biomass and waste sources.
BIOENGINEERED 251
Renowned for its reliable and developed technol­
ogy, B2E is an eco-friendly approach that can
generate green energy from biomass or post-
consumer waste [33,34]. Among the available
B2E technologies, MFC has demonstrated promis­
ing prospects in directly converting biomass to
bioelectricity over exoelectrogens (anodophilic)
microorganisms [15]. In general, the bioconver­
sion of organic matter for bioelectricity production
from MFC could be depicted in Figure 3. The
MFC consists of two chambers, an anode chamber
and a cathode chamber, separated by a proton
exchange membrane (PEM). Further, the exoelec­
trogens are typically immobilized on the surface of
the anode, resulting in a biofilm over the anode
surface to debase the biomass, releasing electrons
and protons, which later be used for bioelectricity
production [35]. The bioelectricity potential and
vitality transformation of MFCs rely significantly
on the efficiency level attained by both the biode­
gradation process and electron transfer [36].
Compared to conventional anaerobic degradation,
MFC manifests greater potential with its higher
energy recovery efficiency from biomass [13].
Typically, conventional anaerobic degradation
produces biogas from biomass, which is suscepti­
ble to 20% entropy lost during its combustive
conversion into useable bioelectricity [8].
Contrarily, such combustive conversion is
252 K. T. X. TONG ET AL.
negligible for MFC as its mechanism involves cou­
pling exoelectrogen metabolism and electrochemi­
cal reaction to convert the chemical energy present
in the biomass into usable electrical energy.
Moreover, MFC can also operate under ambient
temperature, thus offering a higher safety index
and exergy efficiency for bioelectricity genera­
tion [37].
Gebreslassie et al. [13] proved the feasibility of
macroalgae for the co-production of bioelectricity
and hydrogen by applying red macroalgae
Saccharina japonica as substrate in MFC. The
authors revealed that energy recovery of 17.3%
was attained with an optimum power density and
hydrogen production of 1.82 W/m2 and 110 mL/g,
respectively [13]. As a further illustration,
S. japonica substrate in the anode chamber is oxi­
dized by fermentative bacteria under dark fermen­
tation operation to produce hydrogen; however,
carboxylic acids, including acetic acid, butyric
acid, propionic acid, and lactic acid (LA) were
produced simultaneously as the by-products. The
produced carboxylic acids are strongly hydrophi­
lic; thereby, the recovery of carboxylic acids from
fermentation broths requires a complicated
separation process, which hinders their practical
application [13]. Hence, the produced carboxylic
acid molecules [composition: R-COOH] partici­
pated in the anodic reaction and oxidized by exoe­
lectrogens into a reduced form for electrons and
protons production, which can be depicted in Eq.
(1). The released electrons from the anodic reac­
tion are then transferred to the anode through the
exoelectrogen cell membrane, where anode acts as
a terminal for the electron transfer process to the
cathode chamber. At the cathode chamber, the
electrons transported from the anode combine
with the protons, which traverse the PEM, result­
ing in the creation of water molecules in the pre­
sence of oxygen [38]. This reaction completes the
electron flow in the circuit, balancing the charges
caused by the anodic reaction and generating elec­
tric current.
Notably, CO2 is released as a biogenic by-
product of microbial respiration and decomposi­
tion of biomass during the anodic reaction, in
which the exoelectrogens and fermentative bac­
teria in the anode chamber of the MFC oxidize
S. japonica substrate as their energy source. From
Eq. (1), R-COOH compounds are broken down,
releasing biogenic CO2 as an inherent metabolic
by-product, considered a natural carbon cycle
[13]. Interestingly, the released biogenic CO2 can
indirectly help mediate the pH shift between the
two chambers by participating in the carbonate
buffering reactions, shown in Eq. (3) [35].
Okoroafor et al. [14] concluded that adding buffer
salts such as phosphates and carbonates to the
anolyte and catholyte is not economically feasible
for large-scale production and will contribute to
an 80% increment of GWP compared to CO2 as
a buffering agent. Furthermore, an integrated
MFC system for bioelectricity and carbon capture
was developed by Zhang et al. [39] using an algal
biocathode with a layer of biofilm consisting of
photosynthetic algae Chlorella vulgaris. With this
technique, the released biogenic CO2 will be sup­
plied to promote better growth and photosynthesis
of the algae biomass [composition: (CH2O)n],
improving carbon fixation [39]. Eq. (4) depicts
that oxygen is generated as an electron acceptor
by photosynthesis, which later is used in the catho­
dic reaction, creating a sustainable cycle loop.
Thus, MFC aligns with green chemistry and pro­
vides a framework for contributing to achieve the
carbon neutrality goal as they operate under mild
conditions to convert the chemical energy of bio­
mass to electrical energy through microbial meta­
���������!
Anodic reaction: R-COOH + H2O Exoelectrogens 2CO2 + 8H+ + 8e− (1)
+ −
Cathodic reaction: 8H + 8e + 2O2 ! 4H2O (2)
Carbonate buffering reaction: CO2 + H2O $ H2CO3 $ H+ + HCO3− (3)
Photosynthesis: nCO2 + nH2O ! (CH2O)n + nO2 (in the presence of algae) (4)
bolism. The detailed reaction for carbon capture
and fixation is further discussed in Section 3.2.
2.2. Bioplastic
Over the years, petrolic plastic consumption and
production have grown significantly, owing to its
adaptability and affordability, making it ideal for
widespread applications [3]. Global plastics produc­
tion has reached over 360 million tons annually,
dominated by petrolic plastics with seizes

Figure 4. World production of synthetic plastics and bioplastics from 2011 to 2020. Adjusted from [40].
276 million tons and up to 50% of that is for single-
use purposes (Figure 4) [40]. Jem and Tan [41]
reported that 82% of plastic waste from total plastic
production was excluded from recycling, in which
the majority of plastic waste was either heaped in
landfills or cremated, with the remainder spewed
into the ocean as litter. As for the plastic waste
dumped into the oceans, the carbonyl group of pet­
rolic plastic absorbs UV radiation below 280 nm,
making the polymer susceptible to photo-oxidative
degradation, breaking down into petrolic microplas­
tics [40]. Petrolic microplastic contamination has
reached an alarming level as they are ubiquitous
and available for ingestion by aquatic organisms.
These aquatic organisms will suffer from entangle­
ment and starvation as their stomachs become filled
with plastic, leading to death. Moreover, petrolic
microplastics can cause genotoxicity and immuno­
toxicity to marine life as they contain polycyclic
aromatic hydrocarbons [42]. Recent research has
shown overwhelming evidence that microplastics
are in tandem with other toxic chemicals (heavy
metals and organic metals), serving as a vector for
their transport in the environment, which then be
enriched in the food chain to endanger not only
marine life but also human beings [43]. Besides, the
accumulation of plastic waste in landfills is a growing
BIOENGINEERED 253
environmental concern as petrolic plastics take
around a century or more to degrade. During ther­
mal-oxidation degradation of petrolic plastics,
methane and ethylene will be produced and contri­
bute to CO2 emission when exposed to sunlight and
heat, impacting human health and climate [3].
The awareness of the pressing environmental
issue regarding plastic pollution has diverted
attention toward the utilization of alternatives,
such as biodegradable plastic (bioplastic) derived
from a biopolymer. Unlike petrolic plastics for
remaining unchanged for centuries, bioplastics
can be degraded relatively quickly by the action
of microorganisms, namely biodegradation, with
a specific surface degradation rate of 21 µm/year
under specific conditions [44]. Bioplastics can be
broken down into nutrient-rich biomass, CO2,
and inorganic compounds, leaving no toxins and
residue behind. The nutrient-rich biomass can be
converted into fertilizer or utilized as feedstock for
biorefinery purposes, while the CO2 released can
be sequestrated for photosynthetic activity by
Plantae [45]. Thus, CO2 emissions are avoided as
no carbon is involved in the manufacturing pro­
cess of bioplastics. One of the most extensively
researched biopolymers for bioplastics is polylactic
acid (PLA). PLA is a biodegradable aliphatic
254 K. T. X. TONG ET AL.
thermoplastic polyester and has excellent market
prospects, with a range of applications from indus­
trial to civilian use [46]. Briefly, PLA can be
defined as a chain-formed oligomer of LA that
polymerizes by either a Ring-Opening reaction of
lactide or by a Direct Poly-Condensation of high-
purity LA that can be derived from macroalgal
biorefinery [41,47,48].
Its monomer, LA, can be found in either D(-) or
L(+) enantiomeric forms due to an asymmetric
carbon atom, but L-LA is the dominant isomer
for commercialized PLA. Compared to L-LA,
D-LA usage in biomedical applications is skeptical
due to its harmful to human health, especially will
cause neurotoxicity in the human body [49]. This
is because L-LA is the primary endogenous agonist
of hydroxycarboxylic acid receptor 1 (HCA1),
which naturally occurs in living organisms and
the human body has enzymes and metabolic path­
ways specifically designed to metabolize L-LA.
Contrarily, D-LA is not naturally produced by
the human body and the enzymes do not effi­
ciently metabolize D-LA, leading to the accumula­
tion of D-LA in the body, which can disrupt the
normal physiological processes of human beings
[50]. Excessive accumulation of D-LA can result in
acidosis, an abnormal decrease in blood pH, affect­
ing detrimental effects on various bodily functions.
Prolonged exposure to acidic conditions can be
detrimental to cells and tissues. Further, heaping
D-LA can induce inflammation and impede tissue
healing and generation, making D-LA not suitable
for biomedical applications [49]. Hence, good bio­
compatibility is an important reason for the wide
use of poly-L-lactic acid (PLLA) in the field of
biomedicine.
Globally, a nearly 14-fold increment of PLLA
has been produced compared to 2011, reaching
circa 22.72 million tons in 2020 [40]. The drastic
growth in annual PLLA capacity (25% of bioplastic
production, Figure 4) stems from its high mechan­
ical strength and durability properties similar to
petrolic plastics [46]. The mechanical performance
of PLLA is characteristic of glassy polymers with
low deformation and high melting temperature
(160–180°C), which is suitable for different pro­
cessing means, including melt-extrusion molding,
vacuum molding, foam molding, and injection
molding. The good flexural strength (up to 140
MPa) and processing ability of PLLA yield various
plastic products, such as bioplastics, filament for
fused deposition modeling, and fabrics for indus­
trial and civilian use [46,51]. With its biocompat­
ibility properties and approval by the United State
Food and Drug Administration (FDA) for biome­
dical applications, high molecular weight PLLA
has been used to produce non-dismantling surgical
sutures and bone screws, while low molecular
weight PLLA as a slow-release drug-packaging
agent [45]. Casalini et al. [52] manifested that the
degradation of PLLA material by the enzymes
produced by the human body can be used for
drug release. By controlling the metabolism rate
and composition of PLLA material in the capsules,
the therapeutic drugs can quickly target the lesion
site, improve its bioavailability, and reduce its
toxic side effects and toxicity to other tissue
types, leading to a benefit for drug delivery and
as a material for implants structure [52].
To comprehensively compare PLLA with petro­
lic plastics from the point of view of carbon foot­
print, a cradle-to-grave LCA along with
Intergovernmental Panel on Climate Change
method was performed by Morão and de Bie
[53]. The authors revealed that the production
cycle of PLA-based plastic demonstrated lower
environmental burdens than petrolic plastic by
uptaking 1.8 tons of CO2 equivalents per ton of
plastic produced [53]. In addition, Abdul-Latif
et al. [54] revealed that the annual production of
macroalgal-based PLLA should be more than
600 million tons to replace the petrolic plastics
for achieving carbon neutrality, which is calculated
by using carbon emission pinch analysis with total
CO2 emission levels and plastic production in the
view of the year 2016 as base study. Based on the
current PLLA production data, there is an urgent
need to accelerate our efforts to increase macro­
algae-based L-LA production to meet the high
demand for PLLA. Besides diminishing carbon
footprint, Coppola et al. [55] also reported that
the production of PLLA saves circa two-thirds of
the energy required for petrolic plastics produc­
tion, which offers an energy-effective process.
Thus, macroalgal-based PLLA is a promising solu­
tion to defossilize the economy.
In order to meet the global market demand,
large amounts of macroalgal-based L-LA are

essential for the industrial-scale production of
PLLA. L-LA can be derived by chemo-catalytic or
microbial fermentation processes. The chemo-
catalytic process uses petrolic chemicals such as
propionaldehyde and acetaldehyde as feedstock
which may be subject to the potential supply pro­
blem of crude oil and its dramatic price variation.
The drawback of the chemo-catalytic process is
that it involves environmentally unfavorable che­
micals (hydrogen cyanide, sulfuric acid, and
sodium hydroxide) and produces only the racemic
(50:50) mixture of L-LA and D-LA, which is not
desirable for food and biomedical applications due
to the metabolic issues that D-LA may cause.
Further, the racemic mixture is not feasible for
PLA production, typically requiring LA with high
optical purity (e.g. ~99% L-LA or ~ 99% D-LA)
[56]. Then, the microbial fermentation process
outcompetes the chemo-catalytic process for high
optical purity L-LA production as particular
microbes have a specificity of ldhL gene that
secretes L-lactate dehydrogenase (L-LDH)
enzymes, which can only convert pyruvate to
L-LA. Typical microbes employed for L-LA pro­
duction are low guanine and cytosine-content
Gram-positive bacteria, namely, lactic acid bacteria
(LAB) [57]. LAB is a group of microorganisms
that belong to the family Lactobacillaceae and is
characterized by their ability to produce L-LA via
carbohydrate metabolism. Being considered the
macroalgal hydrolyzate composed of mixed rare
sugars, which need to be completely metabolized
for better L-LA conversion yield, the LAB offers an
excellent regulatory mechanism known as carbon
catabolite repression (CCR) for efficient carbohy­
drate metabolism [58].
CCR is the central governing mechanism in
LAB for the regulation of rare sugar uptake,
which ensures cellular resource efficiency by selec­
tively regulating the uptake and metabolism of
favorable carbon sources in the presence of non-
favorable carbon sources, leading to both the
favorable and non-favorable carbon sources can
be metabolized albeit in a different rate [59]. In
brief, CCR in LAB is regulated by different ele­
ments participating in rare sugar transport and
metabolism, including sugar phosphotransferase
system (PTS), enzyme I (EI), phosphocarrier his­
tidine protein (HPr), and catabolite control
BIOENGINEERED 255
protein A (CcpA) acts as the trans-acting repressor
[60]. PTS is a complex transport system responsi­
ble for the uptake and phosphorylation of various
rare sugars in the macroalgal hydrolyzate to
acquire energy and regulate carbohydrate metabo­
lism. The PTS transporters are sugar specific and
phosphorylate the incoming sugar at the expense
of phosphoenolpyruvate (PEP), mirroring the
most suitable carbon sources available in the
hydrolyzate. Notably, the energy for sugar trans­
port is derived from PEP by donating the phos­
phoryl group as an energy carrier through the
phosphotransferase reaction. This reaction con­
verts PEP to pyruvate and transfers the phosphoryl
group to a histidine residue on EI. EI, in turn,
transfers the phosphoryl group to HPr. From the
HPr, the phosphoryl group is transferred to the
permease complex of the LAB cell. Then, the
phosphoryl group is further transferred from the
permease complex to the incoming sugar mole­
cules, resulting in sugar phosphorylation. This
phosphorylation step enhances the diffusion of
sugar into the LAB cell and contributes to its
subsequent metabolism [61]. Further, CcpA is
a regulatory protein that acts as a transcriptional
regulator facilitated in mediating CCR by control­
ling the expression of genes involved in metaboliz­
ing non-favorable carbon sources when the
favorable carbon source is available. The CcpA
will bind to specific DNA sequences called catabo­
lite-responsive elements (cre), which are generally
found in the promotor regions of target genes. The
binding of CcpA to cre sequences modulates gene
expression, leading to activation or repression of
target genes involved in the utilization of non-
favorable carbon sources, which further enhances
carbohydrate metabolism [60]. Along with this
functionality, Ahorsu et al. [62] revealed that the
probiotic-based LAB Bacillus coagulans can simul­
taneously metabolize xylose in the presence of
glucose for L-LA production. Besides, mesophilic
LAB Lactobacillus plantarum also showed clear
diauxic growth in a glucose-mannose-galactose
mixture by attaining a L-LA conversion efficiency
of approximately 65% [47].
Depending on the particular LAB strain and
the specificity of aldolase, the L-LA fermentation
pathway can be divided into homolactic fermen­
tation and heterolactic fermentation process. LAB
256 K. T. X. TONG ET AL.
Figure 5. Simplified illustration of the cellular mechanisms involved in lactic acid production under homolactic fermentation and
self-inhibition in the cytoplasm of Lactobacillus delbrueckii subsp. bulgaricus in anaerobic wort fermentation.
possesses the aldolase enzyme is considered the
homofermentative LAB, which can convert the
carbohydrate-rich hydrolyzate exclusively into
L-LA. Homofermentative LAB includes the strain
from Lactococcus, Streptococcus, Pediococcus,
Lactobacillus, and Enterococcus sp [57,63]. Due
to the high energy yield characteristic that is
beneficial for cell anabolism and cellular growth,
the glucose molecule is the favorable carbon
source for most of the LAB strains. In this per­
spective, glucose molecules will be metabolized
via the Embden-Meyerhof glycolytic pathway by
the homofermentative bacteria. For an insightful
understanding of the Embden-Meyerhof glycoly­
tic pathway, the glucose metabolism by homofer­
mentative LAB, Lactobacillus delbrueckii subsp.
Bulgaricus is illustrated in Figure 5. Glucose
molecules are phosphorylated to glucose-6-phos­
phate (G6P) and transported into the cell by
facilitated diffusion through PTS at the expense
of one adenosine triphosphate (ATP) molecule.
The ATP yield per glucose molecule diffused
inside the cell is 2. Then, the G6P will convert
to pyruvate by glycolysis yielding two molecules
of ATP and one molecule of reduced nicotina­
mide adenine dinucleotide (NADH) per pyruvate
[63]. The overview of the main steps and reac­
tions involved in the glycolysis pathway is
depicted in Figure 6. During the homolactic fer­
mentation process, the G6P is phosphorylated by
the enzyme phosphofructokinase-1, consuming
one ATP molecule, forming fructose-1,6-dipho­
sphate (FDP). The FDP is then cleaved into three-
carbon molecules: dihydroxyacetone phosphate
(DHAP) and glyceraldehyde-3-phosphate (G3P).
DHAP is an isomer for G3P, but only G3P can
directly continue through the next steps of glyco­
lysis. Although both three-carbon molecules exist
in equilibrium, but the equilibrium is ‘pulled’
strongly downward as G3P is depleted. Thereby,
DHAP will convert into G3P by the enzyme triose
phosphate isomerase, ensuring full utilization of
both molecules. The G3P molecules undergo oxi­
dation by the enzyme glyceraldehyde-3-phosphate

Figure 6. Mechanism of glucose glycolysis for lactic acid formation by lactic acid bacteria.
dehydrogenase, resulting in the production of
NADH and the formation of 1,3-diphosphoglyce­
rate (1,3-DPG). NADH carries high-energy elec­
trons that will later be used in ATP synthesis.
Subsequently, 1,3-DPG donates one phosphate
group to the adenosine diphosphate (ADP) mole­
cule for producing an ATP molecule, forming
3-phosphoglycerate (3-PG). For further reaction,
the 3-PG is converted into its isomer, 2-phospho­
glycerate (2-PG). Then, 2-PG loses a molecule of
water via a dehydration reaction catalyzed by
enolase, becoming PEP. PEP is an unstable mole­
cule, poised to transfer its phosphate group to
ADP molecules for ATP generation. As it loses
its phosphate, PEP is converted to pyruvate, the
end product of glycolysis. This reaction is cata­
lyzed by the enzyme phosphoglycerate kinase
[64]. Consequently, L-LA is produced by the
action of L-LDH on the pyruvate. On
a molecular level, the metabolization of pyruvate
by L-LDH is an additional means of NADH recy­
cling. NADH plays a crucial role in the recycling
BIOENGINEERED 257
of NAD+ to maintain glycolysis and the produc­
tion of ATP through the L-LA fermentation,
allowing the continuous breakdown of glucose
and energy production under anaerobic condi­
tions. The recycling pathway for NADH can be
described as follows: (1) During glycolysis, the
NAD+ is reduced to NADH when G3P is con­
verted to 1,3-DPG; (2) During L-LA fermenta­
tion, a hydride ion (H−) from NADH is
transferred to pyruvate for the formation of
L-LA, resulting the regeneration of NAD+. By
regenerating NAD+ from NADH, L-LDH ensures
the availability of NAD+ for the continuation of
glycolysis [65]. However, the produced L-LA has
to be actively transported out of the cell at the
expense of an ATP molecule. This is because of
the fact that at high intracellular pH, L-LA dis­
sociates into lactate and a proton. By maintaining
the intracellular pH and proton motive force, this
proton has to be exported via the plasma mem­
brane proton ATPase at the expense of one ATP
per proton. Once exported, at a low extracellular
258 K. T. X. TONG ET AL.
pH, the L-LA is again present in its protonated
form. In theory, one molecule of glucose can
produce two molecules of L-LA through homo­
lactic fermentation [66].
In contrast to homofermentative LAB, hetero­
fermentative LAB can decompose carbon
sources into a variety of biochemicals, including
L-LA, acetic acid, bioethanol, and CO2. Whereas
CO2 gas detection is a diagnostic test for hetero­
fermentative from homofermentative fermenta­
tion. Heterofermentative LAB includes the strain
from Leuconostoc, Oenococcus, and Weissella sp
[57,63]. The typical reaction pathways of hetero­
fermentative fermentation include the phospho­
gluconate pathway, pentose phosphate pathway,
and phosphoketolase pathway (Figure 6). During
the phosphogluconate pathway, the glucose
molecules are initially phosphorylated into G6P
through the process of PTS and participate in
the glycolysis process, which occurs in the cyto­
plasm of the LAB cell. The G6P is further oxi­
dized by the enzyme glucose-6-phosphate
dehydrogenase, resulting in the production of
one molecule of NADH and 6-phosphogluconic
acid. Through the pentose phosphate pathway,
two half-reactions occur simultaneously: (1)
6-phosphogluconic acid is oxidatively decar­
boxylated by the enzyme 6-phosphogluconate
dehydrogenase; (2) NAD+ is reduced to
NADH. The overall reaction is exergonic, releas­
ing energy that is then used to decarboxylate the
6-phosphogluconic acid, generating CO2 and
ribulose 5-phosphate (Ru5P). The Ru5P mole­
cule is then converted into other pentose phos­
phate sugar, ribose-5-phosphate (R5P) or
xylulose-5-phosphate (X5P), which is catalyzed
by the enzyme phosphopentose isomerase. The
interconversion of Ru5P and R5P allows the
synthesis of essential cellular components,
including nucleotides and coenzymes for cell
propagation. Subsequently, the X5P molecule is
subjected to the phosphoketolase reaction and
cleaved by the enzyme phosphoketolase, result­
ing in the production of two molecules includ­
ing G3P and acetyl phosphate (AcP). Then, the
G3P generated from the phosphoketolase reac­
tion is further metabolized through the usual
glycolytic pathway, similar to the steps in the
conventional Embden-Meyerhof glycolysis
pathway. Whereas G3P will enter the down­
stream steps of glycolysis, leading to the produc­
tion of ATP and L-LA. While the other product
of the phosphoketolase reaction, AcP is decar­
boxylated by the enzyme pyruvate decarboxy­
lase, resulting in the release of CO2 and the
formation of acetaldehyde. Lastly, acetaldehyde
is reduced by the enzyme alcohol dehydrogen­
ase, utilizing NADH as a cofactor. This step
converts acetaldehyde into bioethanol [64]. In
theory, one molecule of glucose can produce
one molecule of L-LA and one molecule of
bioethanol through heterolactic fermentation
process [66]. However, the CO2 release and co-
production of L-LA and other organic acids,
which required additional separation and purifi­
cation processes also hindered the employment
of heterofermentative LAB for microbial fermen­
tation [57]. Therefore, the utilization of homo­
fermentative LAB is preferred over
heterofermentative LAB for L-LA and PLLA
commercial production due to it offering the
high yield, productivity, and optical purity
of L-LA.
3. Microbial fuel cell (MFC)
Contingent upon several cathodic reactions, MFC
diversification has been developed to explore the
particularity in the mechanism of distinct pro­
cesses while enhancing the product output. Multi-
faced applications based on the eventual antici­
pated product reorientated the nomenclature of
MFC as microbial electrosynthesis system (MES),
microbial desalination cell (MDC), electro-
fermentation (EF), and microbial electrolysis cell
(MEC). However, EF is explicitly applied for the
production of a broad range of bioenergy and
biochemicals, potentially achieving higher conver­
sion efficiency (88%) than other MFC systems
(44%) and conventional anaerobic fermentation
(28%) [67]. Thereby, the mechanisms of EF will
be further discussed in the section below.
3.1. Electro-fermentation (EF) – Enhanced
L-lactic acid and bioelectricity production
EF is an electrochemically induced technique that
regulates electron flux in both working electrodes

(anode/cathode) and microbial augmentation,
directing redox reactions toward specific products
at a higher rate [67,68]. In this context, the bioe­
lectricity generated by polarized working electro­
des is not the main energy source but is instead
a product of interest and triggers the fermentation
process driving toward an imbalanced intracellular
oxidation-reduction potential (ORP) condition
[69]. The intracellular ORP is particularly impor­
tant as it controls enzyme synthesis and gene
expression in the electroactive bacteria, thus
impacting the whole metabolic process and path­
way. It can be estimated from the reduced/oxi­
dized nicotinamide adenine dinucleotide
(NADH/NAD+) ratio due to intracellular redox
homeostasis [70]. Variations in the NADH/NAD+
ratio of the system influence the direction of elec­
tron flux, which will be bidirectional and thereby
help resolve the shortcomings of conventional fer­
mentation for desirable product yields. Electron
dispersion at the anode tends to reduce the
NADH/NAD+ ratio, resulting in compensatory
cellular regulation favoring pathways for NADH
replenishment. Subsequently, this mechanism
Figure 7. Basic components of microbial fuel cells and the materials used for structure [36].
BIOENGINEERED 259
further elongated the oxidative and reductive con­
ditions in the EF system and promoted the forma­
tion of ATP molecules for cell division and
anabolism, which results in a higher yield of pro­
ducts. Choi and Sang [71] revealed increased bio­
chemical production yield in response to the
additional NADH through cellular regulation.
Literature related to the virtues for the co-
production of biochemicals and bioelectricity
using EF system showed an increasing trend with
expanding research outputs, leading EF technology
a potential methodology for biorefinery [72,73]. An
overview of the basic components comprising an
EF system is mandatory to understand the signifi­
cant role played by biofilm-producing microbes in
the EF system. The typical EF system is assembled
in a dual-chamber design consisting of an anodic
and cathodic chamber divided by a PEM (Figure 3).
Other designs, including single-chamber and
stacked EFs, are also retrievable from the literature
[74,75]. Figure 7 depicts the tenable materials that
can be employed in the fabrication of the EF sys­
tem. The anodic chamber is made up of one of the
two primary components of EFs, which is referred
260 K. T. X. TONG ET AL.
to as an anaerobic chamber, signifying the critical
role of exoelectrogens and electron mediators in it.
When subjected to an appropriate anaerobic con­
dition, exoelectrogens in the anode function as bio­
catalysts, oxidatively decompose organic substrate
or hydrolyzate into simple molecules or biochem­
icals. This results in the generation of electron-
proton pairs, which are later transferred to the
cathodic chamber through the external circuit and
PEM, respectively. As the electrons travel along the
external circuit, inducing the current flow in the
connecting wire, thereby producing bioelectricity
[76]. Generally, aerating the anodic chamber is
prohibitive, as it could retard the anaerobic perfor­
mance of exoelectrogens in the EF system. This is
because the presence of oxygen can result in the
stripping of dissolved CO2 from the anode cham­
ber, elevating the pH and inducing alkaline condi­
tions. The pH shift may detrimentally affect the
microbial community and electrochemical reac­
tions, hurdling EF performance. Moreover, exces­
sive aeration can lead to mechanical agitation and
disruption of biofilm formation on the anode sur­
face, reducing microbial activity and hindering elec­
tron transfer processes [77].
Contrarily, the cathodic chamber is referred to
as an abiotic chamber, comprising electron accep­
tors for the electron reduction process [36].
Potassium ferricyanide (K3[Fe(CN)6]) is widely
accepted as the electron acceptor, which attracts
and dissipates the electron generated in an anode
by reducing its Fe (III) ion to Fe (II) ion before
reacting with the proton migrated from PEM. In
terms of power generation, K3[Fe(CN)6] is an
excellent electron shuttle, facilitating the transfer
of electrons from the anode to the cathode, which
enhances the electrochemical activity and perfor­
mance of the EF system. This electron transfer
stimulates the metabolic activity of exoelectrogens
by balancing the redox reactions in the system
[78]. Farah et al. [79] revealed that the power
output attained are 8.33 W/m3 and 0.06 W/m3 for
K3[Fe(CN)6] and phosphate buffer as catholyte,
respectively, highlighting potassium ferricyanide
possess superb catalytic activity. However, the uti­
lization of K3[Fe(CN)6] is limited to large-scale
applications due to poor reoxidation, which
demands the catholyte to be frequently replaced.
Further, the long-standing function of the system
can be affected by the diffusion of ferricyanide
ions across the PEM into the anodic chamber,
resulting in aeration anodic chamber, which retard
the metabolic activity of exoelectrogens [80]. Then,
oxygen is employed as an electron acceptor owing
to its availability. Such a process only yields water
as the final product, signifying its positive envir­
onmental benefit, but oxygen possesses limitations
of limited diffusivity (~2 × 10−5 cm2/s) and low
solubility (2–4 mM) in water [81]. Considering
the trend of miniaturization and integration for
future EF systems, potassium permanganate
(KMnO4) is one possible candidate oxidant due
to its characteristics, including high oxidation abil­
ity, large redox potential, and high regenerability.
KMnO4 is a potent oxidizing agent with a high
standard reduction potential that can readily
accept electrons from the anode, which enhances
the electron transfer efficiency in the EF systems
and promotes higher power generation [82].
KMnO4 has been applied successfully to yield an
output voltage of 3003 mV, which conveniently
powers low-voltage appliances [83]. In addition
to the type of electron acceptor, catalyst activity
is also a crucial factor in the performance of the EF
cathode [84]. Giordano et al. [85] further boosted
the power output of the EF system from 70 mW/
cm2 to 115 mW/cm2 by applying 0.1 M sulfuric
acid (H2SO4) as a supporting catholyte. This result
underlined the positive effect of adding H2SO4 as
a supporting catholyte in the EF cathodic chamber
on the catalyst activity and electrochemical output
[85]. The addition of H2SO4 as a supporting elec­
trolyte improves the overall ionic conductivity of
the system for electron transfer between cathode
and anode as H2SO4 dissociates in water to release
hydrogen ions (protons) and sulfate ions. The
presence of hydrogen ions in the catholyte facil­
itates proton conduction from the cathode to the
anode through the electrolyte, enhancing the elec­
trical conductivity and open-circuit voltage. In
fact, proton transfer is essential to the overall
electrochemical reaction occurring in the EF sys­
tem [82]. Besides, the sulfate ions generated from
the dissociation of H2SO4 can act as the ion
exchange sites of PEM due to sulfate ions
approaching the behavior of sulfonic groups in
Nafion®, increasing the mobility of protons
through the membrane [85].

Regarding electrode material selection, chemical
stability, biocompatibility, and conductivity are
desired properties for optimizing EF performance
[86]. Although benchmarked with its conductivity
and arc erosion resistance, copper is not an ideal
electrode material due to its antibacterial features.
During anodic reactions, copper ions released
from the anode will participate in redox reactions
with the protons and generate reactive oxygen
species (ROS), such as superoxide ions and hydro­
xyl radicals. The generated ROS can cause oxida­
tive stress within exoelectrogen cells, damaging
cellular components and disrupting the microbe
cell membrane, leading to leakage of cell contents
and ultimately cell death. Consequently, high cop­
per concentrations can result in oxidative damage
and impede microbial growth [87]. Thereby, non-
corrosive and less harmful stainless steel and
molybdenum steel mesh provide an excellent alter­
native to copper for electrode purposes in the EF
system. Similarly, carbon-based materials, includ­
ing graphite, carbon cloth, carbon felt, and carbon
nanotubes, are among the most widely used elec­
trode materials with high adaptability and excel­
lent electrical conductivity [88]. Other than
conductivity, the choice of electrode materials sig­
nificantly influences the biofilm formation process
and direct electron transfer performance of exoe­
lectrogens to the anode. In EF, the exoelectrogens
or bacterial attachment to and biofilm formation
on the anode surface is crucial for the efficient
biological transfer of electrons between the exoe­
lectrogens and anode [89]. Biofilm is an extracel­
lular polymeric substance (EPS) encased, surface-
adhering microbial community. EPS is a self-
produced matrix of exoelectrogens comprised
polysaccharides, proteins, nucleic acids, and other
biomolecules that surround the biofilm to provide
structural support and protection to the biofilm
community, which is an advantage for exoelectro­
gens’ survival. In theory, the formation of biofilm
on the anode involves several steps: (1) exoelectro­
gens present in the anodic chamber initially come
into contact with the anode surface as anode pos­
sess conductive properties and availability of elec­
tron donors, which is suitable for microbial
attachment; (2) exoelectrogens begin to adhere to
the anode surface via weak and reversible interac­
tions which facilitated by factors such as van der
BIOENGINEERED 261
Waals interactions, hydrophobic interactions, and
electrostatic forces; (3) Upon exoelectrogens
adhere to the anode, they will secrete EPS that
holds the microbial cells together and provides
structural stability to the biofilm; (4) When bio­
film developed, exoelectrogen cells within the bio­
film proliferate and undergo cell division, resulting
in an increment in the population within the bio­
film; (5) Within the biofilm, synergistic relation­
ship develop between exoelectrogens, allowing for
the electron transfer, exchange of metabolites, and
enhanced biofilm functionality; (6) Over time,
channels and void spaces may form within the
biofilm as additional EPS and exoelectrogen cells
are produced, stimulating the movement of meta­
bolic by-products, nutrients, and electron transfer
[90]. In this respect, the preferred characteristics of
anode materials for efficient biofilm formation and
development include good surface charge, rougher
surface, hydrophilicity, and affinity for EPS com­
ponents [89]. Among electrode materials, carbon
felt and carbon nanotubes are the preferred mate­
rial owing to the presence of microscopic irregula­
rities, such as cervices and pores, aggrandizing
structural integrity and biofilm stability.
Furthermore, complementary charge characteris­
tics of carbon nanotubes offer an excellent electro­
static interaction between the charged surface of
the anode and exoelectrogens, facilitating micro­
bial adhesion and subsequent biofilm development
[88]. Nonetheless, the influences of electrode
materials and arrangement on the power output
will be discussed in Section 4.2.
Employing a dual- or single-chamber EF system
relies heavily on the proportion and compatibility
of the final products formed at the counter elec­
trode. Precisely, owing to good scalability and
irreplaceable simplistic design, single-chamber sys­
tems depict greater up-scaling convenience [91].
In most wastewater remediation cases, only an
anodic chamber is mandatory, with the cathode
exposed directly to the air. Hence, the inclusion of
PEM can be excluded from this design, lowering
the manufacturing cost [92]. As shown in Figure 8
(A), the anode and cathode are positioned at each
end of the tube, with only the former covered by
a flat plate, indicating the simplest design for
a single-chamber system. Operationally, the anodic
chamber houses the organic substrate and
262 K. T. X. TONG ET AL.
Figure 8. (a) Single-chamber air-cathode microbial fuel cell reactor [93], (b) single-chamber membraneless microbial fuel cells
operating on the open-air cathode [92], and (c) single-chamber microbial fuel cells operating on stainless steel mesh painted by
acrylic-based graphite [94].
exoelectrogens, while oxygen from the air is con­
stantly supplied to the cathode through diffusion,
which yielded a maximum power density of 1320
± 50 mW/m2 by using sodium acetate as the sub­
strate [93]. Lee et al. [92] came up with an air-
cathode MFC design having the carbon felt anode
inside a cylindrical chamber, while the wet-proof
platinum-coated carbon cloth air-cathode is posi­
tioned on the exterior of the device for wastewater
remediation (Figure 8(B)). The authors revealed
that 82.79% of methylene blue dye in the waste­
water was successfully degraded while simulta­
neously generating bioelectricity with a current
density of 4571.43 mA/m2 over 3.5 h of operation
under simulated solar light illumination [92]. In
another study, a cubic structure single-chamber
air-cathode MFC was developed by applying six
acrylic-based graphite-coated stainless-steel mesh
as anode and six activated carbon-coated stainless-
steel mesh as cathode, which is illustrated in
Figure 8(C) [94]. This study indicated the impor­
tance of surface modification on stainless steel
mesh to increase the overall power output of the
system by 50.22%, yielding a maximum power and
current density of 463.88 mW/m3 and 1991 mA/
m3, respectively [94]. Nevertheless, besides the
aforementioned merits of the air-cathode single-
chamber system, the application of this system is
still hindered by some setbacks. Complexity for
air-cathode manufacturing is considered
a significant setback as diffusion layer or noble
metal catalysts, including platinum and argentum,
are required to enhance the reduction perfor­
mance. The increased complexity and associated
costs can pose challenges for large-scale applica­
tions. Additionally, air-cathode can be degraded
due to several factors, such as biofouling, chemical
reactions, or mechanical stress. Regularly exposed
to oxygen-rich environments, chemical reactions
will take place at the cathode surface to produce
 BIOENGINEERED 263

ROS, leading to the degradation of the cathode
surface and reducing the cathode’s lifespan [82].
Recently, multi-chamber, also known as stacked
EF systems, have been explored as a potential
design configuration for enhanced efficiency in
biochemical production and electrical power capa­
city. Notably, the power capacity can be increased
by connecting multiple individual unit chambers
in either parallel or series configurations. Halim
et al. [86] reported an 87% chemical oxygen
demand removal efficiency with an optimal
power density of 115.94 mW/m2 was attained in
a parallel-wired stacked EF system. In a different
study, a serially stacked EF system comprising
graphite felt as the primary material for working
electrodes was proposed by Kim et al. [95], which
yielded 3.3 V as the maximum voltage, realizing
higher efficiency up to 16.5 times (boosted voltage
from 0.2 V to 3.3 V) compared to single EF system.
Coincidentally, a declination in power output was
discerned over time in both configurations. This
was mainly attributed to ionic short-circuiting and
voltage reversal. With only a single anolyte and
catholyte input, organic overloading could occur
due to inefficient flow distribution. Accumulation
of organic substrate or deposition of non-
conductive materials on the electrode surfaces
can increase internal resistance of the EF system,
leading to electrode fouling and voltage reversal.
This further results in biofilm decay and reduced
the electron transfer efficiency [96]. In view of this,
the conventional configuration, the dual-chamber
EF system is preferred for co-production of bio­
chemicals and bioelectricity. Dual-chambered sys­
tems with membranes are applied when the optical
purity of the bioproducts, such as L-LA and suc­
cinic acid is emphasized [97]. Similar to the pre­
vious genre, dual-chamber EF systems also come
in diverse designs after years of development.
From Figure 9 (D), the anodic and cathodic cham­
bers of the dual-compartment EF system are
assembled in a flat-plate design, in which the elec­
trodes are stacked into a single layer divided by
a PEM. Such design impelled an enhanced proton
transfer with the closely packed electrodes, yield­
ing a maximum open circuit voltage and power
density of 637 mV and 232 mW/m3, respectively
[99]. In addition, Gebreslassie et al. [13] proposed
a H-shape dual-chamber EF system with Nafion®
117 PEM salt bridge completing the circuit and

Figure 9. Diverse configurations for membrane-based microbial fuel cells. (a) Cuboid double-chamber MFC, (b) cylindrical double-
chamber MFC. (c) spherical double-chamber MFC, (d) flat-plate double-chamber MFC, and (E) H-shape double-chamber MFC. In all
designs, the “A” and “C” indicate anode and cathode, respectively [98].
264 K. T. X. TONG ET AL.
sustaining the electrical neutrality of the device
(Figure 9(E)). The authors revealed that the dual-
chamber EF system can be a promising integrated
reactor process for the co-production of hydrogen
and bioelectricity by achieving a hydrogen yield of
110 mL/g and a power density of 182 mW/m2 [13].
Aside from the physical design parameters, the
biocatalyst inoculated to the anode surface also
imparts a significant impact on the bioelectricity
generation potential and biochemical conversion
efficiency of the EF systems. The endowment of
pure or mixed microbial communities for bioelec­
tricity production depends on the system’s archi­
tectural and physiochemical environment,
including electrode size, electrode materials, inter­
nal resistance, and acidity of the system [100].
Thus, the bioelectricity generation potential of
a microbial culture cannot be compared with
each other unless all physical and chemical para­
meters are similar. In the EF system, the capacity
of microbes to decompose the organic substrates is
primarily governed by the composition of the
microbial community and its adaption [101]. The
use of exoelectrogens as biocatalysts is favorable
for generating electricity by organic substrate
degradation [68]. Among exoelectrogen genera
and species, the strain isolated from
Clostridiaceae, Shewanellaceaae, Geobacteraceae,
and Pseudomonadaceae genus, is of great interest
phylum because of their capacity for extracellular
electron transfer (EET) by direct electron transfer
to the working electrode [102–104]. These exoelec­
trogens are usually negatively charged and gener­
ally known as Gram-negative microbes due to the
presence of uronic acids and ketal-linked pyru­
vates in the cell plasma membrane, thereby sup­
plying the anode with positive potential and
divalent cations, including calcium and magne­
sium, which maximizes the scope of biofilm for­
mation on the anode surface by involving
electrostatic interactions [105]. Then, a negative
potential is generated at the cathode to expedite
the reductive reaction with the oxidizing agent by
increased electronegativity in the system [68]. In
terms of direct electron transfer, Clostridium sp.
can catalyze EET to the working electrode by
excreting ferredoxin as endogenous mediators,
while gram-negative proteobacterium belonging
to the Geobacteraceae genus uses pili as conductive
filament for EET [103]. In addition, Shewanella sp.
possesses both EET mechanisms, which use nano­
wires as conductive filaments and excrete ribofla­
vin as endogenous mediators to catalyze
bioelectricity production [106]. Mechanistically,
the metabolism of the organic substrate by exoe­
lectrogen drives the production of NADH within
the cell. For the cell to retain reducing power,
NADH will re-oxidize to NAD+ by abstracting
electrons and biochemicals, such as L-LA, by
using dehydrogenase enzyme and the cytochrome
system comprising menaquinone/quinone pool,
periplasmic proteins, MacA, outer membrane
OmcE protein, and outer membrane OmcS pro­
tein. These cytochrome proteins facilitate the elec­
tron transports by a series of redox reactions
spanning the inner cytoplasmic membrane across
the periplasmic space until the electrons are con­
ducted across the outer membrane to the anode
through outer membrane cytochromes OmcE and
OmcS. Further, the direct electron transfer also
occurs within multilayers of cells through a dense
network of appendages with metal-like conductiv­
ity known as bacterial pili/nanowires. The transfer
is manipulated cell by cell until electrons are
donated to the electrodes [107].
The details of pure microbial cultures used in the
EF systems are provided in Table 1. Through pure
microbial cultures, an insightful alteration in the
process taking place during electron transfer and
the complex behavior of individual species in
mixed microbial cultures can be explored and stu­
died. To date, exoelectrogenic Geobacter sulfurredu­
cens have been gaining attention in the scientific
community for their capacity to generate high
power density [104]. Applying Geobacter strains in
EF systems manifests a significant advantage,
whereby the generated electrons are directly trans­
ferred to the electrode through pili while vying for
the limited surface area on the anode, which yielded
a power density of 325 mW/m2. Theoretically, elec­
trons were transferred to the anode via the mem­
brane-bound redox proteins, with the direct EET
controlling the overall shuttling rate [120].
Furthermore, pure culture with exoelectrogenic
G. sulfurreducens was also applied successfully to
metabolize carboxymethyl cellulose for the genera­
tion of acetic acid with a concentration of 77 mg/L
and bioelectricity with a maximum power density of
 BIOENGINEERED 265

Table 1. A comparison study of pure culture and co-culture for electrofermentation system on L-lactic acid and bioelectricity
production.
Intended product
Working Redox L-lactic acid
Electroactive bacteria strain Biomass/Anolyte potentiala (V) mediator (g/L) Bioelectricity References
Pure culture approach
Shewanella oneidensis Glucose −0.30 Riboflavin - 138,181b [102]
Lactiplantibacillus plantarum Kale hydrolysate 0.20 - 1.13 752.14b [97]
Lactococcus lactis Glucose 0.35 - - 212.00c [73]
Bacillus subtilis Pharmaceutical residual water 0.20 - - 105.00c [108]
Lactobacillus pentosus Diary whey 0.55 Yeast extract - 5.04c [109]
Clostridium cochlearium Glucose 0.19 Thiamine - 5,300.00b [103]
Riboflavin
Bacillus megaterium Activated sludge from wastewater 0.40 Exogenetic - 170.00c [110]
with acetate flavins
Shewanella oneidensis Sodium lactate 0.20 - - 89.40c [111]
Shewanella oneidensis Bamboo fermentation effluent 0.30 - - 578.00c [112]
Lactobacillus bulgaricus Diary whey 0.20 - 19.50 288.12c [113]
Bacillus tequilensis Glucose 0.17 - - 407.96c [114]
Geobacter sulfurreducens Cellulose 0.30 - - 1146.00c [115]
Co-culture approach
d
Bacillus subtilis eSaccharomyces Glucose 0.50 - - 602.00b [116]
cerevisiae
d
Lactococcus latis eLactobacillus Glucose 0.20 - - 415.00c [117]
acidophilus
d
Bacillus tequilensis Glucose 0.17 - - 8,159.27c [114]
e
Pseudomonas aeruginosa
d
Shewanella oneidensis Glucose 0.20 - 7.50 123.41c [118]
e
Engineered
Saccharomyces cerevisiae
d
Shewanella oneidensis eKlebsiella Glycerol 0.15 - - 10.00b [119]
pneumonae
a
Working potential: working electrode potential versus standard hydrogen electrode (SHE) potential.
b
Electric output in terms of current density with SI unit mA/m2.
c
Electric output in terms of power density with SI unit mW/m2.
d
Main electroactive bacteria strain.
e
Supplementary electroactive bacteria strain.

1146 ± 28 mW/m2 in a dual-chamber EF system by riboflavin in the concentration of 100–500 nM

Jiang et al. [115]. In contrast, Shewanella strains are
relatively weaker in the aspect of generating electri­
city from the organic substrate. Evidently, only par­
tial oxidation of sodium lactate into acetate can be
provoked by Shewanella oneidensis, resulting in
a lower overall amount of generated bioelectricity
with a power density of 89.40 mW/m2 after 6 opera­
tion days. This can be attributed to the anaerobic
nature of organic substrate metabolism and the non-
central extracellular feature of the intermediates
[111]. However, Dai et al. [112] revealed that
a higher power density of 578 mW/m2 was attained
using exoelectrogenic S. oneidensis in a dual-
chamber EF system with bamboo fermentation
effluent as the substrate for 8 operation days. The
increment of the power capacity is significantly
affected by the operation period as S. oneidensis
will secrete flavins, including flavin mononucleotide
(FMN), flavin adenine dinucleotide (FAD), and
after 1 week of operation [98]. The excreted flavins
will then act as redox mediators to promote electron
transfer from microbial cells to the electrode surface.
The FAD and FMN are mainly involved in inter­
cellular metabolism by mediating the oxidation of
NADH and reduction of NAD+ to maintain the
imbalanced intracellular ORP. At the same time,
riboflavin is proposed to interact directly with
outer membrane c-type cytochromes as a bridge
between the working electrode and the cell for EET
[121]. The redox mediators can be reduced or oxi­
dized by the exoelectrogens and then be recycled
electrochemically at the working electrode [122].
Indeed, García-Mayagoitia et al. [108] concluded
that the flavins excreted by the Bacillus subtilis strain
and work as redox mediators were shown to
enhance the bioelectricity production in the MFC
system up to 105 mW/m2.
266 K. T. X. TONG ET AL.
In addition to the self-secreted (endogenous)
mediators by the exoelectrogens, Shirkosh et al.
[102] explored bioelectricity production through
EF from a glucose medium by using S. oneidensis
with exogenetic riboflavin. Such endowment
prompted an enhanced bioelectricity capacity and
lifespan of the system, which yielded a maximum
current density of 138,181 mA/m2 with
a prolonged operating duration of 50 days. These
results are nearly 445% better than the values
obtained without exogenetic riboflavin, indicating
that the exogenetic riboflavin is essential for the
survival of S. oneidensis in anaerobic conditions by
offering the ability to access insoluble electron
acceptors [102]. This is attributed to the relatively
slow production rate of endogenous mediators by
S. oneidensis and will only produce limited con­
centrations in the stationary phase when the
microbial cells are lysing [98]. Thus, the endow­
ment of exogenic riboflavin in the culture medium
can boost the electron transfer process by under­
going reversible redox reactions, shuttling between
its reduced (riboflavin semiquinone) and oxidized
(riboflavin) forms. The riboflavin present in the
extracellular environment will interact and bind
with the outer membrane c-type cytochromes of
exoelectrogens, forming a complex called ribofla­
vin semiquinone with an electron attached to it.
Then the riboflavin semiquinone diffuses from the
exoelectrogen cells to the electrode surface. Once
at the electrode surface, the riboflavin semiqui­
none transfers the electron to the electrode, com­
pleting the electron transfer pathway and shifting
back to its oxidized form. This transfer is consid­
ered a direct electron transfer through the interac­
tions of the mediator with the electrode,
facilitating electron exchange [123]. Another
study reported bioelectricity production from acti­
vated sludge via EF using Bacillus megaterium with
exogenic flavins. The optimum power density of
bioelectricity was enhanced by 4.6-fold and
reached approximately 170 mW/m2 compared to
that without flavins addition under similar operat­
ing conditions [110]. Similar results were also
found in the study of Schwab et al. [103] that the
bioelectricity with a maximum current density of
5300 mA/m2 could be generated from glucose by
Clostridium cochlearium isolated from the mouse
gut after 5.2 h of operation duration with the addi­
tion of exogenic thiamine and riboflavin.
To date, most species studied for bioelectricity
generation in EF systems are Gram-negative
microbes. Interestingly, few Gram-positive LAB
strains from Lactobacillus, Lactococcus,
Lactiplantibacillus, and Bacillus genus are recognized
to be both electroactive and able to metabolize a broad
range of carbohydrates to valuable L-LA
[73,97,108,109]. The LAB strains isolated from the
abovementioned genus are considered excellent pro­
ducers of high-purity L-LA as they contain only the
ldhL gene that secretes L-LDH enzymes, which
induces the formation of pure L-LA [124]. In this
regard, Tejedor-Sanz et al. [97] demonstrated that
both rare sugar (xylose and glucose) in the kale hydro­
lyzates can be fermented simultaneously to obtain
a L-LA yield of 1.13 g/L and bioelectricity with
a maximum current density of 752.14 mA/m2 by
using Lactiplantibacillus plantarum at optical density
(OD600) of 1.0 in a H-shape dual-chamber EF system.
Under the same OD600 of Lactobacillus bulgaricus
cells, the EF process for the diary whey was optimized
to achieve the intended product in terms of L-LA yield
of 19.5 g/L and bioelectricity with an optimum power
density of 288.12 mW/m2 [113]. Similarly, an EF sys­
tem with Lactobacillus lactis DLP27 strain as anodic-
respiring bacteria has been explored with
a bioconversion yield of up to 212 mW/m2 from
glucose medium for bioelectricity production [73].
This is mainly due to the fact that L. lactis can excrete
liposoluble quinones (2-amino-3-carboxyl-
1,4-naphthoquinone) to facilitate the EET by connect­
ing the intracellular metabolism of the cell to extra­
cellular redox reactions [125]. Mechanistically, LAB
present in the electron-rich substrate, such as hydro­
lyzate comprising glucose molecules, will manipulate
PTS to transport the glucose molecules to its cyto­
plasm for metabolism reaction. Once the glucose
molecules are at the cytoplasm, LAB will proceed
with either homolactic or heterolactic fermentation
to produce L-LA as the end product. Glycolysis of
glucose molecules drives the production of electrons
within the cell, resulting in an imbalance of ORP level.
For 100% coulombic efficiency, 24 electrons will be
produced per glucose molecule. In order for the cell to
maintain a balance ORP level, it will excrete liposolu­
ble quinones as an endogenous mediator to shuttle

Figure 10. Schematic principle of a microbial fuel cell with lactic acid bacteria as biocatalyst for co-production of L-lactic acid and
bioelectricity [126].
the electron away from the cell to the electrode surface
(Figure 10). In brief, LAB will metabolize the glucose
molecules, abstracting L-LA and electrons. The elec­
trons are then transferred from the LAB cell to the
electron mediator and subsequently to the electrode,
generating an electrical current in the external circuit
of the EF system [97].
Although research is emerging in the pure
microbial culture EF system, all these methods
are extendable to fermenters and electroactive bac­
teria co-culture for supplementary benefits
[118,119]. A comparative study has been reported
on pure culture and co-culture for bioelectricity
production from glucose medium as an anolyte in
the EF process (Table 1). The yield of bioelectricity
in terms of current density was enhanced by
141.92% with co-culture using B. subtilis as an
electroactive bacteria and Saccharomyces cerevisiae
as a fermenter [116]. This is mainly contributed by
the syntrophic interaction among the bacteria
strains, which offers an interspecies electron trans­
fer (IET) between the bacteria, either directly via
conductive pili or indirectly via diffusion of redox
mediator [127]. In this case, B. subtilis excreted
BIOENGINEERED 267
flavins as mediators via respiration to induce the
EET in the anode and reduce the internal resis­
tance of the EF system. Subsequently, S. cerevisiae
transmits electrons through the flavins produced
to reduce the resistance of electron transfer as well
as a direct contact mechanism. Finally, the biopro­
ducts produced by the decomposition of glucose
by S. cerevisiae via metabolism served as an elec­
tron donor and carbon source for B. subtilis. The
bioproduct consumption by B. subtilis in the bio­
film through IET mechanisms facilitates the EF
process and increases the purity of the intended
products [116]. Further, Abdel-Gelel et al. [117]
developed a co-culture EF system by utilizing
a combined LAB strain of L. lactis and
Lactobacillus acidophilus for bioelectricity produc­
tion from a glucose medium. A pure culture EF
system was conducted as a standard reference,
where pure L. lactis and co-culture produced
a power density of 152 mW/m2 and 405 mW/m2,
respectively [117]. The presence of cyclic diguany­
late monophosphate in the intracellular of L. acid­
ophilus stimulates biofilm formation and promotes
L. lactis to produce more liposoluble quinones
268 K. T. X. TONG ET AL.
for EET to minimize the internal resistance
and increase the output voltage of the EF sys­
tem [117].
3.2. CO2 capture and conversion
Associated with the EF reaction in the MFC sys­
tem is the generation of hydrogen (H+) and hydro­
xyl (OH−) ions in the anolyte and catholyte,
respectively, which creates a pH imbalance in the
system as the H+ ions mass transfer is sluggish that
will be accumulated in the anode causes anolyte
acidification [93]. Experiments have shown that
acidic or basic pH significantly decreases the cur­
rent density, voltage efficiency, and resultant
power output by detrimentally affecting the elec­
troactive bacteria metabolism via Le Chatlier’s
principle [128,129]. From an electrochemical per­
spective, pH imbalance also increases concentra­
tion overpotential for substrate oxidation and
potential loss for the whole EF system [129].
Chen et al. [130] revealed that inorganic carbons
(carbonic acid, H2CO3; carbonate ions, CO32-;
bicarbonate ions, HCO3−) produced through the
reduction of nearly 100% CO2 generated during
microbial respiration are indispensable in EF to
offer ionic conductivity and retain stable pH con­
ditions of the electrolyte. The CO2 generated in the
system reacted with OH− ions to form inorganic
carbons, which became the highest concentration
of anions in the catholyte. With an anion exchange
membrane, the inorganic carbons were trans­
ported from the cathode to the anode to retain
electroneutrality and catalyzed the diffusion flux of
anion ions [93].
Aside from being applied successfully for CO2
capture, EF is adapted more toward CO2 fixation
using various mechanisms. In the context of CO2
fixation using EF, the potential differences of the
working electrodes act as a steering force to con­
vert/reduce CO2 to value-added bioproducts due to
CO2 being thermodynamically stable [131]. One
such mechanism is electrochemical CO2 reduction
to bioethanol through biological interventions that
capture and convert CO2 [97]. Besides L-LA, short-
chain carboxylic acid such as acetic acid formation
also occurs due to the degradation of L-LA by the
heterofermentative L. plantarum. The accumulation
of acetic acid and L-LA creates a surge in
maintaining the balance between anolyte and cath­
olyte pH that manifest in the CO2 reduction and H2
formation via the reduction of H+ ions, thereby
promoting bioethanol production in the presence of
H2 as an electron donor by reducing the acetic acid
with cathode poised potential between −0.7 and
−0.9 V [97]. Biogas such as biomethane (CH4) synth­
esis was also perceived upon CO2 reduction in sev­
eral research using EF [132,133]. CO2 reduction
within the EF system toward biogas upgradation
enhances product yield and lowers the process ener­
getics. In general, volatile fatty acids and H2 are
electron donors in converting CO2 to CH4. With
the ability of H2 to implement as a carbon-free
biofuel for the transportation sector, direct IET
mediated by nanowires or pili of electroactive bac­
teria was proposed as an alternative to interspecies
H2 transfer [134]. CO2 is reduced by direct electron
transfer at the catholic chamber at a potential lower
than −0.24 V vs SHE, with 94.14% of the applied
potential converted into CH4 [133]. Methanogens
assist in exploiting in situ produced H+ ions and
electrons as a redox mediator for direct IET coupled
to CO2 reduction toward increased CH4 synthesis
involving less activation energy as a part of the
electrocatalytic activity, which is an added bene­
fit [135].
Alongside the production of bioelectricity, the
production of medium carboxylic acids including
butyric acid and caproic acid was also observed
during the EF system operation using Clostridium
kluyveri as catholic respiration bacteria at −0.6 V
for CO2 fixation [136]. The simultaneous presence
of CO2 and acetic acid in the EF system presenting
Clostridium spp. can lead to longer chain carbox­
ylates via the reverse β-oxidation chain elongation
pathway (Wood-Ljungdahl pathway) by excreting
the acetyl-CoA to promote elongation from acetic
acid (C2) to butyric acid (C4), and then from
butyric acid to caproic acid (C6) [137]. Notably,
butyric acid and caproic acid possess high com­
mercial values and are broadly employed as anti­
microbial agents, feed additives, and flavor
enhancers in the pharmaceutical and food indus­
tries [138]. Each final product has designated
poised potential regulated by the applied potential
for driving the bioelectrochemical synthesis of
bioethanol/biogas/carboxylic acids via CO2 reduc­
tion. EF-MFC integration has depicted
 BIOENGINEERED 269

a significantly increased CO2 capture and conver­
sion toward carbon neutrality in the total biopro­
ducts production by slashing the GWP of the
process [139,140].
4. Microfluidic microbial fuel cells
Advances in microtechnology and growing societal
demand for portable devices that can operate for
extended periods without recharging have paved
a new route for the development of miniaturized
electronic devices and materials for diverse indus­
tries, including environmental, biological, defense,
and clinical applications [141,142]. From that per­
spective, a microfluidic microbial fuel cell (µMFC) is
a promising alternative L-LA production platform
and carbon-neutral energy source for bioplastic
applications and portable microelectronic devices
[143]. The idea of µMFC emerged from the conven­
tional MFC with a total cell volume of 1–250 µL.
Operating such devices in a microenvironment offers
a quick response time to reactants, high surface area
to volume ratio, robustness, and can be well staked
or scaled up [144]. Nevertheless, most quintessential
µMFCs are downsized MFCs, with poor energy effi­
ciency being one of the major setbacks (Table 2).
High internal resistance is acknowledged as the pri­
mary factor of inadequate power output, which is
ascribed to the involvement of the membrane [149].
To realize the real-time feasibility of µMFC, co-
laminar fluid flow arrays are being exploited, where
immiscible anolyte and catholyte flow in the micro­
channel to make µMFC membrane-less which elim­
inates the external influence of inertial forces on the
separation membrane while still consolidating the
advantages of microfluidic flow for power generation
[147]. The common channel configuration of mem­
brane-less µMFC is depicted in Figure 11, whereas
both electrolytes are separated by the diffusional
layer created by laminar flow.
An I-shape microchannel membrane-less µMFC
with a total volume of 50 µL produced a maximum

Table 2. An overview of some common challenges encountered in conventional microbial fuel cell technology.
Main obstacles Description Reference
High internal resistance The involvement of PEMs or separators between the anodic and cathodic chambers introduced additional [145,146]
resistance to proton transport.
Limited scalability Scaling up dual-chamber MFCs is challenging due to the increased complexity and potential issues [111]
associated with maintaining the PEMs.
Low coulombic efficiency The separation of anodic and cathodic chambers by PEM can lead to side reactions or electron losses, [144,147]
reducing the overall efficiency of electron transfer.
Low power density MFCs currently have lower energy conversion efficiency due to electron losses by high internal resistance. [141]
Maintenance and system MFCs require regular maintenance to prevent membrane fouling, electrode degradation and system [141]
complexity failures.
Membrane fouling PEMs in dual-chamber MFCs are susceptible to scaling or fouling attributed to the accumulation of [144,147]
biofilms, salts, or other contaminants, lowering the membrane permeability and hindering proton
transport.
Slow startup and lag MFCs often require a startup period for exoelectrogens or microbial colonization and biofilm formation, [148]
phase leading to a delay in power generation as low cell density is inefficient for electron transfer.

Figure 11. Schematic design of microchannel for membrane-less microfluidic microbial fuel cell: (a) top view and (b) cross-sectional
view [98].
270 K. T. X. TONG ET AL.
power density of 35,294 mW/m2 by S. oneidensis
MR-1, which is circa 200% increment compared to
membraned µMFC. The two streams are mixed by
transverse diffusion alone [102]. In fact, the fluidic
Reynolds (Re) number of the two streams in the
microfluidic chamber, which contrasts the inertial
force to the viscous force, is comparatively small
that ranges between 10 and 100 and is typically
considered as laminar flow [144]. Due to the nat­
ure of the laminar flow, the mixing of both streams
is mainly restricted to a narrow interface and
defined as diffusion across the mutual liquid–
liquid interface between the two streams trans­
verse to the direction flow. The liquid–liquid inter­
face between the two streams in the laminar flow-
based µMFC offers certain advantages over static
membrane µMFC, including (1) promotes convec­
tive transport over diffusive transport so anolyte
crossover can be eliminated as the amount of
diffusion transverse to the direction of flow can
be regulated with high precision by alteration of
the anolyte and catholyte flow rate; (2) eliminating
the water management issue caused by the separa­
tion membrane as it has to be hydrated all times to
facilitate the transport of subatomic atoms; (3) can
operate at elevated temperatures which enhances
the bioelectrochemical reaction due to promote
the microbial kinetics [148].
Owing to their modest size, µMFCs are compa­
tible with several simple microfabrication techni­
ques. Initially, non-polymeric materials including
glass and silicon were utilized as starting materials
for microfluidic device fabrication. Glass materials
have remarkable biocompatibility and possess
superior endurance to harsh conditions (high
acid concentration and temperature) and are
thereby preferred. However, non-polymeric-based
µMFCs are commercially limited due to their
micromachining complexity, high fabrication
cost, and non-optical transparency of silicon
[150]. Hence, polymeric materials such as poly­
methyl methacrylate (PMMA) and polydimethyl­
siloxane (PDMS) have gained considerable
popularity in replacing non-polymeric materials
for µMFC device fabrication due to their design
flexibility, transparency, biocompatibility, durabil­
ity, and low cost [150].
Latterly, some well-developed fabrication tech­
niques demonstrated for lab-on-chip and
electronics applications have been effectively
adapted to µMFC fabrication. Photolithography,
soft lithography, xurography, and paper-based
µMFC are a few types of fabrication techniques
among them. In general, photolithography is
applied to non-polymeric materials in which
a thin film of photoresist (typically SU-8) is spin-
coated on glass or silicon wafer and covered with
an image mask, then exposed to ultraviolet (UV)
light for soft baking. Chemical vapor deposition is
used to remove the exposed layer of photoresist,
resulting in mold [145]. But this technique has
limitations like non-suitability for pattern creation
on non-planar surfaces and the requirement of
expensive equipment. Thus, for polymeric-based
µMFC, soft lithography was established as an
extension of the photolithography technique as it
is flexible and economical [150]. In addition, soft
lithography utilizes a patterned elastomeric poly­
mer as mold, allowing polymers, organic mono­
layers, and gels to be used for microchannel
fabrication [151]. Briefly, a layer of image mask
and the photoresist is coated on the elastomeric
polymer. The masked polymer is then subjected to
UV light, and the unexposed part is removed by
immersing it in the SU-8 developer solvent. The
remaining part is employed as mold, and poly­
meric materials such as PMMA and PDMS are
poured into the mold and rectified to obtain the
intended microstructure [152]. Luo et al. [147] soft
lithographically fabricated a 10 µL Y-shaped mem­
brane-less dual-chamber µMFC using PMMA and
SU-8 photoresist. Using 50 mM potassium ferro­
cyanide as the catholyte and 10 mM lactate as an
anolyte, the authors observed a maximum power
density of 360 mW/m2, with 65% electrochemical
efficiency [147]. The PMMA micromachining
combined with the soft lithography technique
was used to fabricate a 350 µm depth microchan­
nel for dual-chamber µMFC, which yielded
a maximum power density of 343 W/m2 [153].
Paper-based µMFCs have also been developed
to fabricate the microchannel by a paper matrix,
which provides significant advantages. To this
extent, the natural wicking action of paper-based
microchannel offers rapid adsorption of exoelec­
trogen fluid that urges adhesion of exoelectrogen
cells to the working electrode and thereby signif­
icantly reduces the retention time of the

bioelectrochemical reaction [154]. In addition,
paper possesses the capability to flow fluids
through capillary action mechanisms, offering
passive liquid transport, and eliminating the
requirement of external ancillary devices for feed­
ing the electrolyte to the µMFC environment
[155]. Several researches have been conducted to
use the paper-based µMFC approach to develop
membrane-less µMFC. Nath et al. [148] discov­
ered a disposable and inexpensive membrane-less
µMFC fabricated on a Whatman filter paper with
a T-shaped microchannel. With this system, the
devices were shown to hold exoelectrogenic
Escherichia coli for 180 min, producing
a maximum current density of 450 mA/m2, and
a maximum power density of 27 mW/m2.
Rewatkar and Goel [154] proposed a 250 mm2
Y-shaped paper-based microchannel that pro­
duced a maximum power density of 145 mW/
m2. Further, an optimal power density of 240 ±
50 mW/m2 was attained using Whatman, grade
Fusion 5 paper fabricated µMFC. This study
revealed that the paper-based µMFC can produce
similar power output as the µMFC operated with
an external syringe pump using similar working
electrodes and electroactive bacteria strain [156].
4.1. Integrated with EF (Advancements in
microfluidic MFC operations-LLA production and
electricity) − 3D printing technology
Recently, additive manufacturing technology based
on 3D printing has imparted significant advances in
the fabrication and design of complex structures
through the Fused Deposition Modeling (FDM)
technique [146]. This technology offers new poten­
tial for designing the highly personalized structure
and the fabrication of final products through the
sequential deposition of thin layers using the extru­
sion of thermoplastic polymeric fiber [157]. The
rising concurrence of adopting the 3D printing
system over the conventional device fabrication
techniques has resulted in numerous benefits
including fabrication of complex geometry with
exceptional resolution in a short period, customiza­
tion, outstanding design flexibility, and material
economy [158]. With these unique and immense
versatility properties, FDM can expand the range of
novel µMFC and electrode architectures. Freyman
BIOENGINEERED 271
et al. [159] have harnessed the 3D printing technol­
ogy for making bacterial graphene-polycarbonate
(PC) electrodes for direct electron transfer using
electroactive bacteria S. oneidensis ATCC70050 for
lysogeny broth valorization. To confirm and facil­
itate heterogeneous electron transfer, tryptic soy
broth as a growth medium was included in the
printing material for 3D-printed graphene-PC elec­
trode fabrication to enhance the electroactive bac­
terial growth. The experimental data reported that
the 3D printed graphene-PC electrode possesses
lower charge transfer resistance (70.65 Ω) com­
pared to the same material solid electrode (177.1
Ω) and produces a volumetric power density of 8.5
W/m3 attributed to better diffusion and higher sur­
face area of the 3D printed graphene-PC elec­
trode [159].
Moreover, Bian et al. [160] revealed that 3D
printing could offer the precise design of 3D
microporous lattice structured electrodes with
UV-curable resin as printing material. Further sur­
face modification of 3D printed polymeric electro­
des with copper plating could enhance the power
density by 12.3-fold compared to copper mesh
electrodes. Notably, the 3D microporous lattice
structure offers excellent biocompatibility and
a large surface area to S. oneidensis MR-1 for
bioproduct conversion and biofilm development,
while the metal coating facilitates direct electron
transfer [160]. Similarly, a rectangular mesh 3D
printed PLA electrode has been developed to pro­
duce a stable power output (43 ± 1 µW) for almost
840 h [161]. A further example of material extru­
sion applied to µMFC device fabrication was
reported by Rewatkar et al. [141]. In this work,
the authors employed FDM 3D printing to manu­
facture a Y-shaped microchannel. The 3D-printed
microchannel represented the working chamber
for anolyte valorization and catholyte reduction.
The 3D printed microchannel comprised an
enduring and flexible rectangular holder made of
conductive acrylonitrile butadiene styrene (ABS)
based filament. Interestingly, conductive ABS fila­
ment showed better sensitivity and transferability
in electrons; with this advantage, it was selected as
the material for 3D printing of the microchan­
nel [141].
Regarding the employment of FDM technology
for the manufacture of ion exchange membrane
272 K. T. X. TONG ET AL.
Figure 12. 3D printable components of a microbial fuel cell [161].
(IEM), You et al. [161] constructed a 3D printed
membrane using Gel-Lay filament, a filament
composed of rubber elastomeric polymers and
polyamide (Figure 12). The power output from
µMFC with a 3D printed membrane was compar­
able to a commercial proton exchange membrane
(240 ± 11 µW vs 177 ± 29 µW). Intriguingly, poly­
amide possesses strengths of low coefficient of
friction which reduces the internal energy loss
caused by the proton transfer to the cathode
chamber via IEM. Additionally, the polymeric 3D-
printed membrane also resists biofouling due to
good fatigue and impact strength properties [161].
Theodosiou et al. [162] compared 3D printed
membrane electrode assembly with a commercial
IEM. The 3D printed membrane from air-dry clay
materials exhibited twofold higher power outputs
(130 µW vs 66 µW) for 11,760 h. This is attributed
to the groove and emboss structure of the 3D
printed membrane, resulting in a larger surface
area than commercial IEM. Patterned 3D-printed
membranes can exhibit reduced concentration
polarization and improved ion exchange transport
performance while alleviating fouling [163].
4.2. Electrode material and arrangement
The achievable current and power output of µMFC
systems are reliant on the electrode material and
architecture, which directly influences the biofilm
formation, catholyte reduction, and electron
transfer of the system. Inevitably, attaining
a higher power performance µMFC requires
a suitable electrode material with a large surface
area, good biocompatibility, good electron con­
ductivity, low electrical resistance, good surface
hydrophilicity, and chemical stability properties
for microbial adherence [164]. Recently, carbonac­
eous-based material has been extensively used for
the construction of electrodes for µMFC systems
[165]. Nonetheless, the commercial carbonaceous
electrode showed a smooth surface with limited
biocompatibility and electrochemical activity.
Notably, a variety of strategies for carbonaceous
electrode surface modification have been pro­
posed. Chen et al. [166] developed a candle-soot-
coated carbon cloth electrode by inoculating
Aeromonas hydrophila in the MFC system. The
candle soot coating altered the hydrophobic sur­
face of the carbon cloth electrode to hydrophilic
and offers a natural 3D mesoporous ordered struc­
ture for biofilm growth. The modified electrode
exhibited the lowest internal resistance of 619 Ω
with the optimal power density of 19.8 ± 0.2 mW/
m2, which was 48.48% higher than the bare elec­
trode under the same operation conditions [166].
The formation of biofilm on the electrode of
µMFCs presents some significant advantages,
whereby the generated electrons are directly trans­
ferred from the microbial cell to the electrode.
This is attributed to the biofilm matrix providing
a structured environment where the microbial cells

can establish physical contact with the electrode
surface, improving electron transfer efficiency and
power generation. Additionally, biofilm improved
the resistance of microbial cells to toxicity by
functioning as a physical barrier to reduce the
direct contact between toxic substances and micro­
bial cells, exhibiting increased tolerance to toxic
substances compared to planktonic suspension
cells [98]. In another study, Liu et al. [167] chemi­
cally modified carbon paper electrodes with 0.5 M
sulfuric acid for the µMFC system. The authors
revealed that the modified carbon paper displayed
a rougher surface with deep ditches as the sulfuric
acid etched the carbon fibers to increase the sur­
face area of the electrode for the biofilm forma­
tion. The modification reduced the anodic charge
transfer resistance with a maximum volumetric
power density of 235.6 mW/cm3, which was
a 1.58-fold increment than the unmodified carbon
paper [167].
Further, carbonaceous-based nanomaterials
such as carbon nanotubes (CNT) and multi-
walled carbon nanotubes (MWCNT) were modi­
fied to enhance anodic biofilm formation. The
CNT composites can improve cellular adhesion
electrostatically and increase electrode surface
roughness [168]. Bandapati et al. [169] realized
the MWCNT-coated HB pencil graphite
(MWCNT/PG) electrode using the dip coating
method. The performance of MWCNT/PG
showed a higher current density (4,605 µA/cm2)
than the bare PG electrode. Besides, the
MWCNT/PG displayed a more extensive biofilm
of microbial attachment and cyclic volumetry,
which indicated that MWCNT offers good electric
contact between the active site of the microbial
and the PG surface [169]. A reduced graphene
oxide nanosheet-coated carbon cloth anode has
been applied successfully for bioelectricity produc­
tion in the MFC system with a maximum power
density of 10.8 ± 0.19 mW/m2. Reduced graphene
oxide nanosheets with superior electrical proper­
ties and rich hydrophilic functional groups had
established a strong electrochemical performance
in the MFC system [170]. Moreover, the usage of
granular electrodes for MFCs is considered a cost-
effective assay for providing high surface areas,
which is advantageous for achieving high power
output [171]. Activated carbon granules as soluble
BIOENGINEERED 273
electron acceptors attached to G. sulfurreducens
are capable of extracting electrons from acetate
and storing the electrons in the granules. Briefly,
the granular activated carbon behaves like
a capacitor where the protons produced from ano­
lyte oxidation will store in the bacterial biofilm on
the granules along with charge and granules are
discharged when in contact with the current col­
lector. Consequently, during the discharge of gran­
ular activated carbon, protons are also released
along with electrons, leading to localized high con­
ductivity and negligible ohmic losses due to low
electron transfer resistance [171].
Notably, the use of metal electrodes is on the
rise due to possessing good mechanical strength
and higher electrical conductivity compared to
carbonaceous-based electrodes. Evidently, an
interesting outcome was perceived during the use
of the molybdenum electrode. This study found
that the molybdenum electrode produced
a comparable power density of 1296 mW/m2 with
an internal resistance of 62.3 ± 3 Ω, which is a 50-
fold increment compared to the carbon paper
electrode. This is mainly attributed to the appear­
ance of the cleft-like alligatoring surface of the
molybdenum electrode upon electrochemical oxi­
dation, which increases the surface areas for bio­
film adhesion and enhances the mass transfer of
electrons [72]. Meanwhile, molybdenum exhibits
biocompatible properties that enhance biofilm for­
mation and support active microbial propagation,
increasing microbial activity and generating elec­
trons. Indeed, molybdenum is chemically stable
and resistant to corrosion in many physiological
conditions, which does not interfere with the cel­
lular function of microbial cells, promoting cell
viability and electrical stimulation when applied
as electrode material [172]. Similarly, Shirkosh
et al. [102] used a zinc electrode in a µMFC system
cultured with S. oneidensis and reported a current
density of 138,181 mA/m2 at a cell voltage of −0.3
V using a glucose medium. Zinc electrodes exhibit
a higher OCP and current density than most car­
bonaceous-based electrodes in µMFCs inoculated
with S. oneidensis, which could be ascribed to zinc
material possessing a faster ion transfer rate in the
electrolyte than the carbonaceous-based electrode
and a higher reduction standard potential. Further,
zinc-based µMFC could enhance the overall power
274 K. T. X. TONG ET AL.
performance by performing two series of redox
reactions, including (1) redox reaction associated
with zinc oxidation, which releases zinc ions into
the anolyte as a new electrolyte; (2) redox oxida­
tion associated with anolyte oxidation by electro­
active bacteria [102].
However, the life span of metal-based µMFCs is
constrained by the corrosive nature of the metal
materials. In contrast, stainless-steel and surface-
modified metal materials have gained increasing
attention for use as an electrode material for
µMFCs [173]. Stainless-steel exhibits passivation
behavior, which will form a thin oxide layer on the
surface when exposed to oxygen to resist corro­
sion and protect the underlying metal from
further degradation. Moreover, stainless-steel
possesses good electrical conductivity, allowing
for efficient electron transfer between the elec­
trode surface and the electrolyte. This conductiv­
ity facilitates electrochemical reactions at the
electrode–electrolyte interface [94]. Liang et al.
[174] proposed a carbon-coated stainless-steel
electrode by inoculating Geobacter spp. in MFC.
The electrochemical measurement of the modified
electrode exhibited the highest current density of
13 A/m2, which is higher than the MFC conducted
with an unmodified stainless-steel electrode (3 A/
m2) through enhancing bioadhesive and electron
transport rate. It has also been shown that flame-
oxidized wolfram is a more effective anode mate­
rial, and a facile wolfram modification yielded
a power density of up to 1036 mW/m2 at ambient
temperature [72]. A carbon nanofibers-PDMS
coated stainless steel (CNF-PDMS/SS) electrode
has been applied successfully in MFC to generate
a maximum power density of 19 mW/m2 after 16
days of operation. Besides achieving higher power
density output than unmodified electrodes, CNF-
PDMS coating can enhance the corrosion resis­
tance of stainless steel by reducing its corrosion
rate from 367 µm/yr to 31 µm/yr [173]. Moreover,
nickel nanostructure modification was performed
to enhance anodic biofilm formation on the nickel
foil electrode. Designing the µMFC using nickel
nanostructure led to a higher volumetric power
density of 343 W/m3 with an additional enhance­
ment of 67% in the surface power density com­
pared to the unmodified nickel electrode. The
increased surface area due to the employment of
nickel nanostructures on the anode side could
increase the density of attached electroactive bac­
teria to the anode surface by enhancement of
carbon source accumulation, thereby elevating
the anolyte valorization and improving the mass
transfer [153].
Aside from the electrode material selection, the
diffusion layer existing over the electrodes also
substantially influences the fuel cell output.
Increasing the electrode surface area while assum­
ing a homogenous diffusion layer could increase
the overall power performance. Nevertheless,
increasing the surface area of a single electrode
pair µMFC system simultaneously increases the
thickness of the diffusion layer, attributing to
electron mass transfer limitation and a decrease
in power density [175]. Kim and Chang [176]
resolved this concern by investigating the effect
of the electrode diffusion layer on MFC power
generation. Using an immobilized inoculum
source collected from a sludge tank in the
Gwangju sewage treatment plant on the anode
as bioanode, they revealed that splitting a single
platinum-coated carbon cloth bioanode into nine
simple bioanode while retaining the electroactive
area and distributing them by some fixed distance
fostered an increase in power output by a factor
of 30%. In conclusion, the use of multiple smaller
bioanodes and increased distance between the
bioanodes could enhance the bioadhesive rate of
electroactive bacteria while preventing an increase
in the thickness of the diffusion layers [176].
A similar report was also put forward by Li
et al. [177] wherein a multi-layer porous electrode
consisting of five single-layer porous carbon
paper electrodes was implemented in the vana­
dium µMFC system for bioelectricity production
through continuous electrolyte replenishment.
The system displayed a 1294% increment in
power output with 120% lower internal resistance
under a multi-layer flow-through porous elec­
trode configuration. This could be attributed to
the use of a multi-layer electrode which offers an
uniform reaction rate and diffusion layers distri­
bution in the porous electrode to cope with oper­
ating conditions resulting in a superior cell
performance [177].
In another study, Jiang et al. [111] integrated
six flow-through µMFCs in an array into

Figure 13. Schematic illustration and fabrication process for the origami array-type µMFC. (a) Schematic representation with dimension (mm),
(b), (c) folded structure in 3D view, and (d) overview of origami array-type µMFC realization on 3D printed platform [178].
a sandwich of two acrylic sheets. Operating the
µMFCs, potassium ferricyanide and S. oneidensis
MR-1 culture medium driven independently
into catholyte/anolyte, the authors revealed 56
µA of total current with a 16.4-fold increase in
volumetric density at 765 µA/cm3 and 4.3-fold
decrease in current generation response time to
achieve 80% of the peak output current than
a single µMFC system [111]. A novel origami
array-type µMFC for bioelectricity generation
was developed by Rewatkar et al. [178] with
a horizontally arrayed 1 cm2 manganese oxide
nanoparticles coated carbon anode and 1 cm2
carbon cathode (Figure 13). The µMFC yielded
a maximum power density of 15.9 µW/cm2 and
OCP of 0.53 V. Further electrochemical analysis
revealed that this integrated flow-through µMFC
configuration improved interfacial nutrient
assimilation to the bacterial colonization,
BIOENGINEERED 275
biocompatibility, and direct electron transfer
rate of the system.
5. Circular biorefinery approach- Microfluidic
MFC/bioplastic closing the loop
The circular biorefinery model has been employed
to enhance the reuse and recycling of current
biomass-derived wastes to produce new bioenergy
and biochemicals using environmentally friendly
techniques. Such the ‘take-make-recycle’ circular
model offered a beneficial way to maintain con­
tinuous growth of the quality of life and economy
by controlling the valorization of finite resources
[179]. µMFCs can be promising circular biorefin­
ery models as they can operate for biochemical
production and energy recovery by valorizing var­
ious biogenic materials and wastes [154]. In this
context, abundance macroalgae are a promising
276 K. T. X. TONG ET AL.
Figure 14. Schematic diagram of circular biorefinery and bioeconomy of microfluidic microbial fuel cell for bioelectricity and
bioplastics production.
feedstock for developing the circular biorefinery
model. The microbial reactions involved in the
macroalgae hydrolyzate acting as a substrate in
µMFC generate electrons and protons which can
later be used for the co-production of bioelectricity
and L-LA along with CO2 sequestration from
organic matters present (Figure 14). Although
CO2 is released into the anodic chamber during
microbial reactions, that can be reused in the loop­
ing system for macroalgae re-growth and as
a buffer medium for the system, making the
µMFC a carbon-neutral technology. Compared to
the conventional biorefinery model, µMFC con­
sumes significantly lower energy rather than gen­
erating bioelectricity with EF reactions [70].
It is crucial to recognize that the macroalgal
biomass upstream bioprocessing may significantly
govern the range of downstream intended biopro­
ducts. Moreover, depending on the targeted bio­
product of interest, selecting the most flawless
approach or combination of approaches is para­
mount [10]. The interconnected structures of
macroalgal cell walls are structurally and chemi­
cally more heterogenous than lignocellulosic mate­
rials as they are a polyphyletic group, which can be
deciphered via the dynamic chemical diversity that
presences between Rhodophyta, Phaeophyta, and
Chlorophyta. Generally, macroalgal cell walls typi­
cally consist of a fibrillar skeleton material, known
as cellulose and an amorphous enmeshed phyco­
colloids (carrageenan, agar, ulvan, glucuronan, or
alginate) unique to each taxa to form a double
fibrillar layer, which ultimately requires an effec­
tive hydrolysis assay to gain access to the internal
cell membrane. This involves either conventional
(chemo-catalytic and bio-catalytic) or innovative
hydrolysis assay which disrupts the macroalgal
cell wall constituents, followed by hydrolysis of
polysaccharides into rare sugars [11]. The use of
chemo-catalytic hydrolysis involving sulfuric acid
(H2SO4) was explored by Hessami et al. [180] to
enhance the extricated rare sugar (glucose and
galactose) yield to 39.42% from red macroalgae
Gelidium elegans.
From the economic standpoint of the macro­
algal biorefinery, phycocolloids have frequently
been the primary target for recovery due to
being commercially valued [181]. Park et al.
[182] and Alfonsín et al. [33] investigated the
extraction of alginate and carrageenan,

respectively, and assessed the appropriateness
of the remaining residues as feedstock for rare
sugar extraction using chemo-catalytic hydroly­
sis assay. Park et al. [182] identified that 40% of
alginate can be extracted from Laminaria japo­
nica, and 107.5 g/L of glucose was successfully
extricated post hydrochloric acid hydrolysis.
Similarly, Alfonsín et al. [33] suggested that
a biorefinery approach using Eucheuma denti­
culatum for the production of carrageenan and
rare sugars could be commercially viable and
yielded 19.7% of extricated rare sugars corre­
sponding to 0.30 g/L of glucose from the carra­
geenan-free residue through acid hydrolysis.
Notably, unfavorable chemo-catalytic hydrolysis
conditions could result in undesirable by-
products including 5-hydroxymethylfurfural
(HMF) and formic acid via degradation of
extricated rare sugars, which is a side reaction
of chemo-catalytic hydrolysis that is unable to
surpass completely. These by-products will
diminish the fermentation efficiency to produce
the carbon-based end-point products, such as
L-LA by hindering the protein synthesis and
damaging the DNA of the fermentative bacteria
[183].
As an alternative to chemo-catalytic hydrolysis,
bio-catalytic, known as enzymatic hydrolysis with
the utilization of enzymes to expedite the cleavage
of macroalgal cell wall constituents and polysac­
charides is preferred as this assay is environmen­
tally benign [184]. The use of cellulase isolated
from the halophilic bacterium Vibrio parapaemo­
lyticus was explored by Hebbale et al. [185] to
enhance rare sugars (glucose and rhamnose) extri­
cation from green macroalgae Enteromorpha
intestinalis. The authors revealed that enzymatic
hydrolysis using cellulase from
V. parapaemolyticus alone was sufficient to com­
plete the saccharification of E. intestinalis with
rare sugar yields of 90.59% and no 5-HMF forma­
tion. This is mainly due to cellulose isolated from
V. parapaemolyticus is composed of thermostable
and acidic endoglucanase, which shows higher
catalytic efficiency on acid-sensitive intermolecu­
lar glycosidic bonds cleavages compared to basic
BIOENGINEERED 277
endoglucanase [185]. In fact, the cellulase from
V. parapaemolyticus contains the lysine decarbox­
ylase (cadA) genes that will degrade the lysine
content in the macroalgal biomass to maintain
the optimum pH conditions (pH 3–8) of the sys­
tem, thereby extricated rare sugars yield can be
enhanced simultaneously by aggrandizing the
enzyme activity [186]. Further, Rocher et al. [24]
performed the enzymatic hydrolysis of brown
macroalgae Ecklonia maxima using yeast
Saccharomyces cerevisiae and justified that the
yield of extricated glucose could be significantly
improved from 29% to 66% by genetically modi­
fied yeast with laminarinase-encoding genes from
Trichoderma viride and Rasamsonia emersonii.
This is attributed to the S. cerevisiae cannot pro­
duce sufficient of its native laminarinase-like
enzyme for effective hydrolysis of laminarin [24].
Although high rare sugar yields can be observed,
this assay is constrained by the extended retention
times of hydrolysis ranging between 1 and 5 days
and high production costs [24,185]. Thus, the use
of enzymatic hydrolysis usually implies with pre­
treatment approach to reduce the retention times
of the process which is indicated as a two-step
hydrolysis assay and considered not viable from
the economic perspective [184].
To improve the setbacks of conventional hydro­
lysis assays, eco-friendly and innovative hydrolysis
assays are increasingly developed, including
microwave-assisted hydrolysis and ozonolysis
[12,187]. Microwave-assisted acid hydrolysis has
been employed successfully to extract galactose
from E. denticulatum carrageenan under 120°C
with an operation duration of 25 min [12]. The
authors concluded that 50.7% of the galactose
recovery rate, corresponding to 27.9 ± 1.69 g/L of
galactose can be achieved along with a low 5-HMF
of 0.74 ± 0.18 g/L from the E. denticulatum carra­
geenan with the involvement of 230 W microwave
power [12]. Cao et al. [188] further applied higher
microwave power (1900 W) to enhance the acid
hydrolysis of Gracilaria lemaneiformis under the
optimized temperature of 180°C with 0.2 M H2
SO4, and the extricated rare sugars (galactose and
glucose) yield reached up to 73.3% with
a retention time of 20 min, which is sixfold lesser
278 K. T. X. TONG ET AL.
compared conventional acid hydrolysis assay.
They revealed that the superficial heat transfer
environment offered by microwave irradiation to
the macroalgae biomass can improve the extri­
cated rare sugars yield and retention time of the
process whilst simultaneously limiting the forma­
tion of 5-HMF [188]. The use of microwave irra­
diation reduced the retention time and by-
products formation, which paths a new route for
the hydrolysis of macroalgal biomass for the cir­
cular biorefinery; however, bottlenecks associated
with the high-power consumption for microwave
heating at the targeted temperature on
a commercial scale were raised.
In contrast, the ozonolysis assay has been widely
applied as a lignocellulosic pretreatment process
for delignification. However, it has not been exten­
sively explored as a hydrolysis assay for macroalgal
biomass as they are less recalcitrant due to a lack
of lignin complexes [189]. The ozonolysis assay
utilizes ozone (O3) to treat the biomass cell walls
and selective carbohydrate degradation at ambient
temperature and pressure with minimal effects on
rare sugars, leading to lower yield in 5-HMF gen­
eration in the extricated rare sugars tenable for
fermentation. As an electrophilic and triatomic
molecule, O3 is highly reactive toward the elec­
tron-rich cell wall matrix and carbohydrates due
to an electron deficiency in terminal oxygen dur­
ing resonance [190]. The reactions of O3 with
carbohydrate molecules involve an initial electro­
philic attack, resulting in the formation of carboxyl
and carbonyl groups followed by hydroxylation of
the carbonyl groups, which increases electrophilic
substitution reactivity of the compounds for gly­
cosidic bond cleavage [191]. Although O3 is an
efficient oxidizing agent (E0 = 2.07 V, 25°C), O3
alone is relatively ineffective in oxidizing cellulose
surfaces [189]. Thus, Tamilarasan et al. [187]
explored hydrothermal ozonolysis to improve
rare sugar recoveries from green macroalgae
Chaetomorpha antennina. Compared with the
control case (ozonolysis alone), total rare sugar
recovery rates and energy ratio increased 2.1 and
2.4 times with water as a hydrolysis solvent. This
attributed to treatment with O3 in an aqueous
medium that generates a reactive radical known
as hydroxyl through the formation of superoxide,
which reacts with the cell wall matrix resulting in
random cleavage of hydrogen bonds to extricate
carbohydrate contents. In addition, soluble low
molecular weight compounds, mainly organic
acids including acetic acid and formic acid, will
be released during ozonation, resulting in
a decrease in the acidification of the solution that
can expedite the cleavage of acid-sensitive glycosi­
dic bonds [187]. Further, Sulfahri et al. [192] sig­
nificantly improved total rare sugar extricate yield
in red macroalgae Kappaphycus alvarezii from
0.16 g/g to 0.52 g/g with ozone-assisted enzymatic
hydrolysis. Hence, ozone-assisted hydrolysis assay
is preferred for rare sugar extrication in terms of
high hydrolytic efficacy and low energy consump­
tion, which allies with the criteria embedded
within the circular biorefinery concept.
Anaerobic fermentation (AF) is the indispensa­
ble sequential stage in the circular biorefinery loop
for the valorization of extricated rare sugars or
glycolytic intermediates toward L-LA production
using acid-tolerant LAB. Among thousands of
strains of identified LAB, Bacillus spp. and
Lactobacillus spp. are the prominent microbial
that have been perceived as the most crucial con­
tributing to beneficial effects in L-LA fermentation
using carbon-rich rare sugars as substrate.
A typical superiority of these LAB strains for
L-LA production is offering strength to growth
under high temperatures up to 50°C, utmost resis­
tance to contamination, and less stringent nutri­
tional requirements [193]. Wu et al. [194]
concluded that 7.02 g/L (48.48%) of L-LA can be
attained during the fermentation of acid-modified
Ulva lactuca hydrolyzates using 10% (v/v)
Lactobacillus plantarum under batch mode at
37°C for 24 h. Under the same inoculum density,
the fermentation process for acid-modified
E. denticulatum cellulosic residues was optimized
to achieve a L-LA yield of 98.6%, corresponding to
14.02 g/L of L-LA (Chai et al., 2021). Lin et al. [47]
also explored the mixed microbial culture of com­
bined L. acidophilus and L. plantarum strains for
L-LA fermentation from red macroalgae Gracilaria
sp. hydrolyzates, finding remarkable L-LA titer
enhancements by merely 30% with L-LA yield of
64.72% compared to monoculture. This may be
due to the probiotic bacterium L. acidophilus will
excrete proteinaceous bacteriocins including lacto­
cin B, acidocin A, and acidocin B during the L-LA

fermentation as nitrogen sources to stimulate
L. plantarum strain growth [195]. Tong et al.
[12] further enhanced the L-LA titer by employing
various inoculum densities of Bacillus coagulans at
37°C for 14 h and revealed that the L-LA conver­
sion yield was improved from 19.93 to 22.49 g/L
with an increment of 88.62% when 4% (v/v)
B. coagulans was applied compared to only 2%
(v/v). In conclusion, a higher volumetric produc­
tivity of L-LA and a shorter metabolization rate of
LAB can be achieved by employing a high inocu­
lum density culture as it offers a rapid fermenta­
tion process between LAB and rare sugars [12].
Apart from the generation of intended biopro­
ducts, the successful reduction of waste within
a biorefinery process is a covetable goal that
ought to be achieved by adopting green technol­
ogies to incorporate within the cascading biorefi­
neries. Lin et al. [118] accomplished this during
their research which showed the viability of gen­
erating bioelectricity from residues of glucose and
glycolytic intermediates that remained post L-LA
production. The study demonstrated this inte­
grated biorefinery assay of L-LA and bioelectri­
city production in a MFC system using
a combined bacterium strain of S. oneidensis
and engineered S. cerevisiae for the EF process.
L-LA yield of 7.50 g/L was attained from the
glucose medium, after which the residues were
utilized as substrates in MFC to generate bioelec­
tricity. The output voltage and power density
obtained from this MFC system were 355 mV
and 123.41 mW/m2, respectively [118]. Tejedor-
Sanz et al. [97] concluded that MFC is
a sustainable CO2 sequestration platform, where
the electroactive bacterium Lactiplantibacillus
plantarum can convert CO2 released during
L-LA fermentation into a variety of value-added
bioproducts (acetic acid, ethanol, and bioelectri­
city). Further, Shirkosh et al. [102] enhanced the
bioelectricity generation by downscaling the MFC
into microfluidic environments, finding
a markedly technological advantage by generating
higher power density output 35,294 mW/m2 com­
pared to MFC. These findings revealed that
µMFC is an emerging waste-to-chemical/energy
platform for CO2 fixation and multiple carbon-
BIOENGINEERED 279
end point bioproduct generation from a single
substrate, indicating a significant opportunity
for developing a circular biorefinery model.
In the circular biorefinery model, the L-LA pro­
duced will be further processed into biodegradable
PLLA polymer for bioplastic application via
a Ring-Opening lactide reaction or a Direct Poly-
Condensation of L-LA [41]. The end-of-life for
such bioplastics can be recycled or biodegraded
using microorganisms. PLLA-based products can
be subjected to chemical recycling. In detail, che­
mical recycling is a depolymerization process
using mild acids or solvents. Then, the depolymer­
ized components can be repolymerized to form
valuable products [196]. In addition, Adhikari
et al. [197] studied the biodegradation of PLLA-
based products. The authors reported that the
fragments components of the PLLA polymers via
biodegradation can be utilized as substrates for
either anaerobic fermentation or EF to exploit
the untapped potential [197]. The energy recovery
from the biodegradation process can be used
further for the production of power or heat
[198]. Thus, a close-loop circular biorefinery
model is demonstrated.
6. SWOT analysis of microfluidic microbial
fuel cell toward bioelectricity and bioplastic
production for circular biorefinery
Technology Transfer is essential to move technol­
ogy from research labs toward commercialization.
Thus, the strengths, weaknesses, opportunities,
and threats (SWOT) analysis of a raised technol­
ogy product must be conducted to identify its
future prospects. An appropriate SWOT analysis
would be beneficial to transfer the obtained scien­
tific findings to industrial- or pilot-scale applied
technology. In the following sections, the possibi­
lity for the transfer of µMFC technology to fruition
will be analyzed regarding the strengths of this
technology, technological limitations, challenges,
and future prospects.
6.1. Main strengths and weaknesses of
microfluidic microbial fuel cell
The most vital strength point of µMFC is the
possible use of organic substrate or wastewater as
280 K. T. X. TONG ET AL.
a fuel source to initiate biocatalytic environmental
remediation and co-production of biochemicals
and bioelectricity. Hence, this unique property
provides a sustainable approach for the processing
of multiphase waste treatment and biomass utili­
zation while reducing CO2 emissions, which is
accorded with the UN Sustainable Development
Goals [175,199]. On account of its modest size,
µMFC can be easily scaled up or down, offering
versatile applications, including bioenergy produc­
tion, wastewater remediation, biosensors, and
remote power generation, and enabling integration
into lab-on-chips devices [147]. Further, the com­
pact nature of microchannels in µMFC minimizes
diffusion limitations by increasing the surface-to-
volume ratio of the system, which promotes mass
transfer capabilities regarding electron and nutri­
ent transfer between the microbes and electrodes.
This allows for improved accessibility of microbes
to the substrates, enhancing the reaction kinetics
and power generation [153]. The elimination of
PEM in the µMFCs is an economically sustainable
strategy that simultaneously reduces the internal
resistance attributed to proton transport, improv­
ing the efficiency of the µMFCs. Moreover, µMFCs
possess the ability for multiplexing and paralleliza­
tion, allowing simultaneous operation of multiple
microchannels or units, which enables higher
throughput and increased power output [152].
Because of the precise microscale features, the
fabrication of µMFCs can be technically challen­
ging and require specialized equipment and mate­
rials. Achieving reliable and reproducible
fabrication processes for µMFCs can be time-
consuming and costly. However, despite having
high mass transfer capabilities, µMFCs are still
hurdled by mass transfer limitations. The limited
volume microchannels in µMFC can overload the
substrate [147]. Besides, µMFC may require inte­
gration with external components, including
pumps, monitoring systems, and sensor, which
contribute to the complexity and challenges in
terms of system reliability, compatibility, and over­
all performance. Additionally, establishing long-
term operational stability and performance consis­
tency of µMFs can be challenging. This is attrib­
uted to the performance of µMFCs relying on
forming and maintaining biofilms on the electrode
surface. The intricate fluidic conditions, limited
space, and potential for biofilm degradation are
the several factors that made biofilm control
a difficult process [148]. Lastly, scaling up
µMFCs from lab-scale to practical applications
while retaining performance and cost-
effectiveness is limited due to mass production
and system integration [167].
6.2. Challenges and future prospects
As conventional MFCs are a well-established tech­
nology and have been extensively studied with
a mature research base and developed protocols,
this could limit the public perception and accep­
tance of µMFCs. Furthermore, the involvement of
intricate designs, specialized fabrication techni­
ques, and complex fluidic dynamics in the study
of µMFCs raises challenges regarding operation
and maintenance, which may also hinder their
widespread adoption [200]. Besides, recent studies
on µMFC have been made without fully employing
biofilm engineering and fabrication techniques
that are specifically designed to enhance µMFC
performance significantly. Consequently, the
development of 3D printing technology for anodes
with biofilm engineering has enormous potential
for supporting the development of high-
performance µMFCs in the future, as they possess
the ability to enhance EET to stimulate power
generation by several orders of magnitude [160].
Immobilization of exoelectrogens in the mesh-like
electrodes can also be considered a potential
advancement to control and maintain biofilm
growth. Regarding microscale features, µMFC
offers opportunities to develop miniaturized bioe­
nergy devices suitable for portable and on-site
power generation in resource-limited or remote
settings. Then, the study of µMFC can be inte­
grated with lab-on-chips systems, allowing for
simultaneous energy generation and biosensing,
enabling multifunctional devices. To expand the
market opportunities and adoptions, researchers
should tailor µMFC design for specific applications
such as biomedical devices or environmental mon­
itoring systems, exposing the usage of µMFCs.
Moreover, µMFC competitiveness can be further
increased by understanding the thermodynamics
of EET at the microscale and maximizing the gen­
eration of all available high-value components

from the single substrate through cascading bior­
efinery. The abovementioned prospects guide
future work toward a full understanding of the
high performance of µMFC system development
as a viable technology.
7. Conclusions
This review delineated the in-built potential of
macroalgae in terms of bioproducts recovery and
simultaneously diminishing the carbon footprint
with a sustainable perspective. There are several
pathways for the current sustainable energy
transition. MFC technologies provide
a potential approach for cascading biorefinery
to exploit the co-production of bioelectricity
and biochemicals from the organic substrate
that could replace conventional sources of pet­
rochemicals. Bioelectricity and L-LA generated
from the metabolism of exoelectrogens or LAB
can be maintained to provide a reliable contin­
uous source of renewable energy and bioplastic
production. Nevertheless, the commercialization
of MFC is a hurdle associated with insufficient
power generation. Currently, investments in cir­
cular biorefinery are focused on using µMFC
system to enhance the power output and achieve
a better conversion yield of L-LA. It promises to
be the most potential biorefinery model to
address the futuristic circular biorefinery with
more innovation in the near future.
Appropriate economic, technical, and scientific
strategies in a multi-disciplinary approach can
lead researchers to pursue a sustainable algae-to-
chemical/energy system by addressing the circu­
lar biorefinery goals and bridging the research
gaps between waste remediation and product
recovery.
Disclosure statement
In accordance with Taylor & Francis policy and ethical
obligation as a researcher, we are reporting that Pau Loke
Show is one of the editors of Bioengineered. We have
disclosed those interests fully to Taylor & Francis, and
we have in place an approved plan for managing any
potential conflicts arising from the publication of this
manuscript.
BIOENGINEERED 281
Funding
This study was funded by the Fundamental Research Grant
Scheme (FRGS/1/2019/TK02/CURTIN/03/2) from the
Ministry of Higher Education (MOHE) Malaysia, Curtin
Malaysia Higher Degree Research (CMHDR) Grant and
Curtin Malaysia Postgraduate Research Scholarship
(CMPRS) from Curtin University Malaysia.
Data availability statement
The data that support the findings of this study are available
from tan.s@curtin.edu.my.
Credit authorship contribution statement
Kevin Tian Xiang Tong: Writing-original draft, Project
administration, Data curation. Inn Shi Tan: Supervision,
Conceptualization, Writing-Reviewing, and Editing. Pau
Loke Show: Investigation and Validation. Henry Foo Chee
Yew: Supervision, Validation. Man Kee Lam: Investigation.
Mee Kee Wong: Investigation and Validation.
ORCID
Kevin Tian Xiang Tong http://orcid.org/0000-0001-6687-
3961
Inn Shi Tan http://orcid.org/0000-0003-1901-8211
Henry Chee Yew Foo http://orcid.org/0000-0002-7831-
9105
Pau Loke Show http://orcid.org/0000-0002-0913-5409
Man Kee Lam http://orcid.org/0000-0002-5517-1072
Mee Kee Wong http://orcid.org/0000-0003-4470-0386
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