Bioresource Technology 394 (2024) 130206

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Enhancing biophotovoltaic efficiency: Study on a highly productive green

algal strain Parachlorella kessleri MACC-38
Nia Z. Petrova *, Tünde N. Tóth , Prateek Shetty , Gergely Maróti , Szilvia Z. Tóth
Institute of Plant Biology, HUN-REN Biological Research Centre, Szeged, Temesvári krt. 62, H-6726 Szeged, Hungary

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

• Parachlorella kessleri MACC-38 generates

exceptionally high electric current.
• P. kessleri MACC-38 grows sustainably in
the presence of redox mediator
ferricyanide.
• Electricity produced by P. kessleri
MACC-38 is largely dependent on
photosynthesis.
• P. kessleri MACC-38 exhibits uncommon
exoelectrogenic properties.

A R T I C L E I N F O A B S T R A C T

Keywords: Biophotovoltaic (BPV) devices are a potential decentralized and environmentally friendly energy source that
Exoelectrogenic activity harness solar energy through photosynthesis. BPV devices are self-regenerating, promising long-term usability. A
Bio-photoelectrochemical cell practical strategy for enhancing BPV performance is to systematically screen for highly exoelectrogenic algal
Ferricyanide
strains capable of generating large electric current density. In this study, a previously uncharacterized green algal
Photosynthesis
strain - Parachlorella kessleri MACC-38 was found to generate over 340 µA mg− 1 Chl cm− 2. This output is
approximately ten-fold higher than those of Chlamydomonas reinhardtii and Chlorella species. The current pro­
duction of MACC-38 primarily originates from photosynthesis, and the strain maintains its physiological integrity
throughout the process. MACC-38 exhibits unique traits such as low extracellular O2 and Fe(III) reduction,
substantial copper (II) reduction, and significant extracellular acidification during current generation, contrib­
uting to its high productivity. The exoelectrogenic and growth characteristics of MACC-38 suggest that it could
markedly boost BPV efficiency.

1. Introduction convert solar energy into electrical current (Wey et al., 2019). However,
unlike the conventional photovoltaic manufacturing process, which
Biophotovoltaic (BPV) devices, like traditional photovoltaics, often involves the use of toxic and carcinogenic substances (reviewed by

* Corresponding author.
E-mail addresses: petrova.nia@brc.hu (N.Z. Petrova), tunde.toth.biol@gmail.com (T.N. Tóth), shetty.prateek@brc.hu (P. Shetty), maroti.gergely@brc.hu
(G. Maróti), toth.szilviazita@brc.hu (S.Z. Tóth).

https://doi.org/10.1016/j.biortech.2023.130206
Received 20 October 2023; Received in revised form 12 December 2023; Accepted 12 December 2023
Available online 18 December 2023
0960-8524/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
N.Z. Petrova et al. Bioresource Technology 394 (2024) 130206

Tawalbeh et al., 2021), BPV devices employ inherently safe biomaterials cupric reductase described in C. reinhardtii (Hill et al., 1996). Therefore,
for light energy harvesting and charge separation. These biomaterials a systematic screening of a diverse range of microalgal species and a
include intact photosynthesizing organisms, isolated photosynthetic thorough understanding of their exoelectrogenic mechanisms are crucial
membranes, or pigment-protein complexes (Bombelli et al., 2011). In a for advancing BPV technologies.
BPV system, electrons derived from the biological component reduce an Screening of green microalgal strains from the Mosonmagyaróvár
anode and are subsequently transferred to a cathode where H+ and/or Algal Culture Collection (Ördög et al., 2013; Horváth et al., 2023) per­
O2 are reduced to H2 and H2O respectively (Wey et al., 2019). formed in this study rendered a highly exoelectrogenic strain – MACC-
Intact photosynthesizing organisms present several advantages over 38, belonging to the Parachlorella kessleri species. Strain MACC-38
the use of isolated photosynthetic complexes. These include longer demonstrated a significantly higher capacity for FeCN-mediated cur­
operation times, the ability to generate electric current in both light and rent production than the model green alga C. reinhardtii and other
dark conditions, CO2 consumption, self-repair capabilities (McCormick P. kessleri strains. Interestingly, the exceptional exoelectrogenic prop­
et al., 2011; Bombelli et al., 2011, 2022) and the potential to couple erties of MACC-38 could not be attributed to RBO and FRE activities, but
power generation with biomass production (Huang et al., 2015). were at least partially due to copper reductase(s) activity. In addition,
The biological processes that contribute to bioelectricity production extracellular acidification was found to have enhancing effect on FeCN-
by photosynthesizing microorganisms largely depend on the nature of mediated current production.
the anode-organism interface. Artificial membrane-permeable com­
pounds, such as quinones, can extract electrons from the photosynthetic 2. Materials and methods
electron transport chain, thereby facilitating electron transfer between
chloroplasts and the BPV anode. However, this approach faces chal­ 2.1. Algal cultures
lenges related to quinone partitioning within cellular compartments,
cellular toxicity, and a rapid decline in photocurrent, which question its Strain MACC-38 was obtained from the Mosonmagyaróvár Algal
long-term applicability (Longatte et al., 2018; Sayegh et al., 2021). Culture Collection (Széchenyi István University, Hungary, Horváth
Alternatively, the interaction between photosynthetic organisms and et al., 2023). P. kessleri strains 27.87, 211–11g and 211-11c were pur­
BPV anodes can be based on the inherent ability of biological cells to chased from the Culture Collection of Algae at the University of
reduce their environment. This phenomenon, known as exoelectro­ Göttingen (SAG, Germany). C. reinhardtii CC-1883, CC-503 and CC-124
genesis, involves the export of electrons through various mechanisms. strains were obtained from the Chlamydomonas Resource Center (Uni­
The exoelectrogenic activity of biological cells can be employed for versity of Minnesota, USA).
transfer of electrons to the BPV anode in a direct or a mediated manner. Algal cultures with a starting cell density of 0.5 × 106 cells ml− 1 were
The direct reduction of the anode has been demonstrated for various grown in Tris-acetate-phoshate medium (TAP) in 100 ml Erlenmeyer
green algae and cyanobacteria (McCormick et al., 2011; Herrero-Medina flasks on a rotatory shaker at 120 rpm, under continuous white light
et al., 2022). While extensive research has been conducted on the mo­ with intensity of 80–90 µmol photons m− 2s− 1 provided by fluorescent
lecular mechanisms, growth conditions, and subcellular structures that light tubes (Sylvania luxline plus). All experimental procedures unless
facilitate photocurrent production in cyanobacteria (Gonzalez-Aravena explicitly stated otherwise were carried out with algal cells harvested in
et al., 2018; Wey et al., 2021), the exploration of the direct electric the beginning of exponential growth phase (3rd day after culture
current production in green microalgae remains limited. Moreover, initiation).
previous reports point out that green microalgae produce negligible In a subset of experiments, the algal cultures were grown in acetate-
power density in the light in BPV setups with direct electron transfer free Tris-phosphate (TP) medium. For this, after two days of growth in
(McCormick et al., 2011). TAP medium, MACC-38 cells were washed twice in fresh TP or TAP
The mediated (indirect) interaction with the BPV anode involves the medium and re-suspended in the respective media. The effect of acetate
use of exogenous plasma membrane (PM)-impermeable compounds was assessed after 24 h of growth in TP or TAP medium.
such as potassium ferricyanide (K3[Fe(CN)6]), FeCN) (Bombelli et al.,
2011; Laohavisit et al., 2015; Anderson et al., 2016; Shlosberg et al., 2.2. Genome sequencing
2021). Using the natural exoelectrogenic properties of microalgae for
FeCN-mediated current production not only yields promising power Total DNA (nuclear, mitochondrial and chloroplast DNA) from pel­
outputs but also maintains cell viability (Bombelli et al., 2011; Wang leted algaal cells was isolated by the DNeasy Plant Mini Kit (Qiagen,
et al., 2021), making it a preferred strategy for photocurrent generation. Germany) according to the instructions of the manufacturer. Paired-end
FeCN is a highly potent electron mediator capable of shuttling fragment library was prepared using the NEBNext® Ultra™ II DNA Li­
electrons from various PM redox proteins (Luthje et al., 2013). The brary Prep Kit for Illumina (Cat.Num.: E7645L). Paired-end fragment
occurrence of redox proteins in PM is a strongly species-specific feature, reads were generated on an Illumina NextSeq sequencer using TG
making the choice of photosynthesizing microorganism a pivotal factor NextSeq® 500/550 High Output Kit v2 (300 cycles). Primary data
in the productivity of BPV. Anderson et al. (2016) reported that in the analysis (base-calling) was carried out with Bcl2fastq2 software
model green alga Chlamydomonas reinhardtii, electric current production (v2.17.1.14, Illumina).
is primarily linked to the activity of the PM NADPH oxidizing protein
respiratory burst oxidase (RBO1), which is responsible for extracellular 2.3. Genome assembly and annotation
superoxide generation (O•- 2 ). On the other hand, green microalgae have
multiple species-specific exoelectrogenic mechanisms beyond RBO- The raw reads were trimmed to remove adapters and drop reads
dependent superoxide formation that have yet to be investigated in shorter than 30 bases using Trimmomatic (v0.39, Bolger et al., 2014).
BPV technology. Previous studies suggest that iron acquisition mecha­ Additionally, all low-quality bases were trimmed using a sliding window
nisms in green algae involve the reduction of Fe(III) ions by PM ferric of 5 bases and an average quality score of 20 across the 5 base sliding
reductase (FRE), which can also reduce FeCN (Lynnes et al.,1998; Allen window. All the filtered reads were assembled with SPAdes (v3.11.1,
et al., 2007). In Chlorococcum litorale, ferric reductase activity is signif­ Prjibelski et al., 2020). Contigs belonging to organelle genomes were
icantly upregulated by iron deficiency combined with extremely high identified by Blast (v2.12.0, Camacho et al., 2009) against reference
CO2 concentration (Sasaki et al., 1998a, 1998b). In the halotolerant Chlorophyta chloroplast and mitochondrial genomes downloaded from
species Dunaliella salina, FeCN reduction capacity is correlated with a NCBI. These contigs were subsequently removed to continue with
Na+ extrusion process (Katz and Pick, 2001). Another potential annotation of the nuclear genome. Any contig with length smaller than
contributor to PM redox processes relevant for current production is the 600 bases were also removed from further analyses.

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Nuclear genome annotation began with repeat identification. Re­ µM DCMU for 20 min in the dark prior to CA experiments.
peats in the assembled contigs were identified and masked using In a subset of experiments, CA measurements were performed at
RepeatModeler (Smit and Hubley, 2008–2015) and RepeatMasker (Smit various pH. For this MACC-38 cultures were washed and incubated in TP
and Hubley, 2008–2015). Structural gene models were predicted using medium buffered with 20 mM MES (pH 5) or 20 mM Tricine buffer (pH 7
BRAKER (v2.1.6, Brůna et al., 2020). A total of 112,824 proteins were or 9) for 5 min.
downloaded from NCBI using “txid35461[Organism:exp]” tag.
Organelle genomes were assembled using GetOrganelle (v1.7.6.1, 2.8. Enzymatic assays
Jin et al., 2020). Circularized organelle genomes (both chloroplast and
mitochondria) were annotated on GeSeq. For determination of FeCN reduction rate algal cultures were settled
Finally, identification of candidate genes involved in modulating down and re-suspended in Phosphate-buffered saline (PBS), pH 7.4. Cell
electricity production such as RBO and FRE were identified by manually suspensions with Chl concentration of 0.05 mg ml− 1 were supplied with
curating a database of RBOs and FREs from Chlamydomonas and Chlor­ 1 mM FeCN (200 mM stock) and placed at 150 µmol photons m− 2s− 1. To
ella species. These reference sequences were downloaded from Uniprot. follow FeCN reduction in time, samples were collected every 10 min for
Orthologues for all these genes were identified using OrthoFinder 30 min and immediately centrifuged. The reduction of FeCN in the su­
(v2.4.0, Emms and Kelly, 2015), with MUSCLE (v3.8.31, Edgar, 2004) as pernatant was evident by the decrease in absorption at ΔA420-650nm.
the alignment program. FeCN concentration was determined using extinction coefficient 1.03
mM-1cm− 1. The FeCN reduction rate was defined as the slope of the
2.4. Growth curves in the presence of potassium ferricyanide linear fit of FeCN concentration in time.
The ability of algal cells to produce extracellular O•−
2 was assessed by
Suspension cultures of the four P. kessleri strains were started with the formation of water soluble formazan upon reduction of 3′-{1-
cell density 0.5 × 106 cells ml− 1 in TAP medium. On the second day after [(phenylamino)-carbonyl]-3,4-tetrazolium}bis(4-methoxy-6-nitro)ben­
inoculation, FeCN was added to the suspension cultures at concentra­ zenesulfonic acid hydrate (XTT) by O•− 2 (Sutherland and Learmonth,
tions of 0.5, 2 or 5 mM. The growth progression of the algal cultures was 1997). Following re-suspension of the algal cells in TP medium, cell
monitored by measuring cell density using a Luna-FL Dual Fluorescence amount corresponding to 0.1 mg Chl(a + b) ml− 1 was incubated with
Cell Counter (Logos Biosystems, South Korea). 100 µM XTT under 150 µmol photons m− 2s− 1.
Plasma membrane ferric and cupric reductase activities were eval­
2.5. Chlorophyll concentration uated according to procedures described respectively by Lynnes et al.
(1998) and Weger (1999). These colorimetric methods are based on the
Chlorophyll (Chl) concentration of the algal suspensions was deter­ ability of bathophenanthroline disulphonic acid (BPDS) and bath­
mined spectrophotometrically after pigment extraction with 99 % ocuproine disulphonic acid (BCDS) to form stable colored chelates with
methanol (Porra et al., 1989). the reduced forms of iron and copper – Fe(II) and Cu(I), respectively.
Prior to performing these enzymatic assays, P. kessleri cultures were
2.6. Chlorophyll a fluorescence induction washed and re-suspended in TAP medium without trace elements and
ethylenediamine tetraacetic acid (EDTA); the Chl concentration was set
For the Chl a fluorescence measurements, the algal cells were dark- to 0.05 mg Chl(a + b) ml− 1. P. kessleri cultures were incubated with
adapted for 10 min. Dark-adapted algal cells were deposited onto BPDS or BCDS for 20 min at 150 µmol photons m− 2s− 1.
Whatman glass microfibre filters (GF/B). Chl a fluorescence induction
transients were recorded by HandyPEA (Hansatech Instruments Ltd., 2.9. pH measurements
UK). FV/FM values were calculated as follows: FV/FM=(FM-F0)/FM,
where F0 is the minimal Chl a fluorescence and FM is the maximum Chl a For detection of pH changes occurring upon illumination with
fluorescence intensity. moderate light (150 µmol photons m− 2s− 1) and upon the addition of 5
mM FeCN, the P. kessleri cultures were re-suspended in unbuffered TP
2.7. Chronoamperometric measurements medium with a cell density corresponding to 0.05 mg Chl ml− 1. pH re­
cordings were taken every 5 min with Orion 3-star pH meter (Thermo
Electric current was measured over time by eight-channel potentio­ Scientific, USA).
stat multiEmStat3 (PalmSens BV, The Netherlands) operating with
screen-printed electrodes (type AC1.W4.R1, BVT Technologies, Czech 2.10. Statistical analyses
Republic) with Ag/AgCl reference electrode (RE), and graphite anode
and cathode. The area of the anode was 0.79 mm2. The chro­ All presented data are average of at least three independent experi­
noamperometric (CA) curves were recorded with 0.5 V bias potential, ments, with one or two biological replicates in each. Statistically sig­
based on preliminary experiments. Prior to CA measurements, 3-day-old nificant differences to the respective controls were established in
algal cultures were pelleted down and re-suspended in fresh TP medium. OriginPro 9.0 Software applying one-way ANOVA with Holm-Sidak
Chl concentration was adjusted to 0.1 mg Chl(a + b) ml− 1 (corre­ post-hoc test at p < 0.05.
sponding to approximately 30–40 106 cells ml− 1 in all P. kessleri strains).
Preliminary experiments demonstrated that, at this specific Chl con­ 3. Results and discussion
centration, MACC-38 achieves its maximum current density in the
presence of 2 mM FeCN. Any potential limitations caused by the medi­ 3.1. Identification of Parachlorella kessleri MACC-38
ator molecule were eliminated by using a concentration of 5 mM FeCN
in all CA experiments. A 130 µL volume of the algal culture was carefully Algae-based BPV devices offer significant advantages in BPV
applied as a droplet onto the screen-printed electrodes. The electric research, such as the ability to convert light energy into electrical cur­
current was then measured for a duration of 1400 s, either in darkness or rent combined with self-regeneration, which promise potential for sus­
at different light intensities ranging from 150 to 2000 µmol photons tained use. However, to achieve commercial viability, considerable
m− 2s− 1. improvements in the efficiency of these algae-based BPVs are needed.
The effect of photosynthetic electron transport inhibitor DCMU (3- One of the promising approaches that could lead to BPV efficiency
(3,4-dichlorophenyl)-1,1-dimethylurea) on current production was improvement is the selection of highly productive algal strains.
determined after incubation of algal cultures (0.1 mg Chl ml− 1) with 50 The Mosonmagyaróvár Culture Collection (Széchenyi István

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University, Hungary) consists of several hundreds of microalgal strains MACC-38 showed the highest productivity. Strain 27.87 yielded an in­
and has been previously proven to be a valuable resource of algal strains termediate value of 148 µA cm− 2 mg− 1 Chl after approximately 23 min,
with biotechnological applicability (Ördög et al., 2013, Horváth et al., while strains 211-11c and 211–11g produced the lowest maximum
2023). Based on the results of an initial larger scale screening for easily current densities of 93 µA cm− 2 mg− 1 Chl and 86 µA cm− 2 mg− 1 Chl,
cultivatable and robust strains with a high biomass yield (similarly as respectively (Fig. 1A). Thus, all tested P. kessleri strains, especially
performed by Ördög et al., 2013, Horváth et al., 2023), a set of green MACC-38, exhibited a greater capacity for FeCN-mediated current pro­
microalgal strains belonging to the Chlorella and Parachlorella genera duction than the C. reinhardtii strains. Interestingly, the original CA
was selected for further electric current production experiments (see curves show that the electricity production by MACC-38 did not reach a
Supplementary material). During this initial screening, a green algal plateau during the CA measurements lasting 23 min (Fig. 1B). In
strain, MACC-38, exhibiting an exceptionally high potential for electric extended measurements lasting 40 min, MACC-38 achieved a maximum
current production was identified. current density of approximately 800 µA cm− 2 mg− 1 Chl (data not
However, the taxonomic identification of this strain using conven­ shown).
tional loci (the nuclear 18S and ITS2 genes) was uncertain, prompting us The existence of diverse microalgae-based BPV designs makes it
to conduct a more precise phylogenetic analysis (see Supplementary difficult to compare the capacities of different algal strains and species to
material). Therefore, chloroplast genes were utilized for better phylo­ serve as the photo-sensitive part of a BPV device and therefore
genetic identification of MACC-38. Specifically, the rbcL, petA, psaB and comparative analysis of electricity yields should take into account fac­
atpA genes were extracted from the annotated chloroplast genome of tors such as contact between algal cells and the electrodes, the use of
MACC-38 and cross-referenced with the non-redundant nucleotide electron mediators, electrode materials, culturing conditions, etc.
database maintained by NCBI. Remarkably, these genes were found to Among the species that have been exploited in BPV current production
be 100 % identical to reference sequences from Parachlorella kessleri. in similar settings to ours, only the high-salinity tolerant species D. salina
showed higher FeCN-mediated current than MACC-38 (Shlosberg et al.,
2021). D. salina was observed to generate 700 µA cm− 2 mg− 1 Chl after
3.2. Electric current production by Parachlorella kessleri 10 min in the presence of FeCN and this increased to 950 µA cm− 2 mg− 1
Chl when FeCN-mediated current measurement was preceded by incu­
To highlight the exceptional ability of P. kessleri MACC-38 to bation with NADP+ (Shlosberg et al., 2021). While these results are
generate FeCN-mediated current, it was initially compared with three indeed valuable, it is worth mentioning that they were achieved under
widely used wild-type strains of the model green alga, C. reinhardtii. The specific conditions of high salinity, which naturally enhances electrical
comparison was conducted under conditions similar to those previously conductivity, and under extremely high light intensity. The latter, while
used to assess the bio-electricity generation capabilities of various green effective in this context, could be potentially harmful for the cells.
microalgal species (Anderson et al., 2016; Shlosberg et al., 2021). In the The electric current densities obtained with MACC-38 in a FeCN-
followings, the electric current output obtained in the light is referred to mediated BPV system were significantly higher than the productivity
as the total electric current, and the light-induced current is the calcu­ reported in previously published studies employing closely related
lated difference between the total and dark-induced current. Chlorella species. For instance, C. vulgaris cells, when treated with
Remarkably, MACC-38 produced a maximum total current density of boronic acid and incorporated into a redox polymer, were reported to
344 µA cm− 2 mg− 1 Chl after 23 min, which is 7–13 times higher than produce a maximum current density of 5 µA cm− 2 (Herrero-Medina
that generated by the CC-503, CC-1883, and CC-124 C. reinhardtii et al., 2022). Similarly, a photosynthetic optical resonator containing
strains. These strains produced current densities of 50 µA cm− 2 mg− 1 Chlorella sp. yielded a current density of 44.2 nA cm− 2 (Roxby et al.,
Chl, 26 µA cm− 2 mg− 1 Chl, and 38 µA cm− 2 mg− 1 Chl, respectively 2020). Immobilized Chlorella sp. generated a maximum current density
(Fig. 1A and B). These findings agree with those of Anderson et al. of 6.78 mA m− 2 (or 0.68 µA cm− 2) under light provided by a LED source
(2016), who reported similar values using BPV devices with the CC-503 optimized to match the absorption spectrum of the photosynthetic
and CC-1883 Chlamydomonas strains and FeCN as an electron mediator. apparatus (Ng et al., 2020). These values are orders of magnitude lower
After 18 h of current production, they recorded a current of 800 µA mg− 1 than what was achieved with the MACC-38 P. kessleri strain character­
Chl using an anode surface of 15.2 cm2, which corresponds to a current ized in the current work.
density of approximately 52 µA cm− 2 mg− 1 Chl.
To provide a more objective basis for comparing the exoelectrogenic
activity of P. kessleri, and particularly MACC-38, the electric current 3.3. Growth of Parachlorella kessleri in the presence of potassium
production of three randomly selected P. kessleri strains from the SAG ferricyanide
culture collection (27.87, 211–11g, and 211-11c) was tested under
identical conditions (Fig. 1A). Among the four P. kessleri strains tested, For an algal species to be effective in sustained and long-term BPV

Fig. 1. Electric current production by P. kessleri and C. reinhardtii strains. A) Maximum electric current density normalized to Chl(a + b) content. The graph shows
mean values ± SE, with n = 3. B) Representative chronoamperometric curves of MACC-38 and CC-503 strains recorded in darkness or at light intensity of 150 µmol
photons m− 2s− 1. Statistically significant differences among total, dark, and light current values are denoted by different letters, as determined by ANOVA with Holm-
Sidak post-hoc test at p < 0.05.

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current production, it is essential that the species not only has a strong with increasing FeCN concentration, both on days 3 and 5 (Fig. 2E and
ability to reduce the BPV anode (or electron carrier), but also that the F).
anode material (or electron carrier) does not have a detrimental effect While the addition of 0.5 and 2 mM FeCN resulted in a slight ac­
on cell viability. In light of this, the growth capacity of the four P. kessleri celeration in the growth of strains 211–11g and 211-11c (which was
strains was evaluated in the presence of varying concentrations of FeCN statistically significant for the 211-11c strain), by the 5th day, both the
– specifically, 0.5, 2, and 5 mM (Fig. 2A, B, C and D). The FV/FM ratio, a treated and untreated cultures reached similar cell densities (Fig. 2C and
widely accepted measure of photosynthetic efficiency and a compre­ D). The addition of 5 mM FeCN did not impact the growth of these
hensive indicator of the overall physiological state of algal cultures (Tan strains (Fig. 2C and D). However, their FV/FM values were substantially
et al., 2019), was evaluated at two time points: 24 and 72 h after the decreased at 2 and 5 mM FeCN, with this effect being more pronounced
addition of FeCN (corresponding to the 3rd and 5th day of cultivation, on day 5 (Fig. 2E and F).
respectively) (Fig. 2E and F). Interestingly, these observations differ from those reported for the
The impact of FeCN on culture growth showed both concentration- closely related species C. pyrenoidosa, where FeCN was found to enhance
and strain-specific characteristics. Specifically, MACC-38 growth photosynthetic activity, alleviate light stress, reduce reactive oxygen
remained essentially unaffected across all tested FeCN concentrations species formation, and increase oxygen evolution by PSII (Wang et al.,
(Fig. 2A), with the exception of the 2 mM FeCN concentration that 2021).
resulted in a slight increase in the growth rate (Fig. 2A). The 2 and 5 mM A key distinction between this experimental setup and CA mea­
FeCN treatment caused only a marginal decrease in the FV/FM ratio on surements is that during electric current measurements, FeCN is re-
both the 3rd and 5th days (Fig. 2E and F). Conversely, the growth of oxidised at the anode and is thus predominantly present in its
strain 27.87 was significantly inhibited at all applied FeCN concentra­ oxidized form. However, during prolonged incubation of algal cells with
tions (Fig. 2B), with a progressive decrease in FV/FM ratio correlating FeCN, it is reduced to ferrocyanide (see section 3.5.) - an effect easily

Fig. 2. Impact of various FeCN concentrations on the growth and photosynthetic activity of four P. kessleri strains. A-D): Growth curves of MACC-38, 27.87, 211–11g,
and 211-11c strains, respectively. The graphs show mean values ± SE, n = 6. E-F): FV/FM values of P. kessleri strains grown in the presence of various FeCN con­
centrations for 24 h (day 3, E) and 72 h (day 5, F), respectively. The graphs show mean values ± SE, n = 18. Different colors in the graphs represent different FeCN
concentrations, as indicated in the legend. Asterisks of different colors denote statistically significant differences between the treatment signified by the respective
color and the control. Statistically significant differences were determined by ANOVA with Holm-Sidak post-hoc test at p < 0.05.

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observable by the visible discoloration of growth media containing eliminated approximately two-thirds of the light induced current pro­
MACC-38 and FeCN. Nonetheless, with the exception of strain 27.87, duction (Fig. 3A).
these results suggest that FeCN-mediated current production does not When MACC-38 cells were grown in acetate-free (TP) medium for 24
exert a strong toxic effect on P. kessleri, either due to changes in cellular h prior to CA measurements, a decrease of approximately 30 % was
redox state or as a side effect of FeCN. observed in both total and light-induced current, while the dark current
Microalgal BPV devices, based on cyanobacterial species have remained unchanged compared to cultures grown in TAP medium
already been demonstrated to be able to support the operation of mi­ (compare Fig. 3A and B). In the presence of DCMU, the light-induced
croelectronic devices (McCormick et al., 2011; Bombelli et al., 2022). current was completely suppressed in photoautotrophically grown
However, green microalgae have not been tested in long-term operating cells (Fig. 3B). This result confirms that the light-induced current
BPV devices yet. The absence of a detrimental effect from FeCN, coupled generated by MACC-38 originates mainly from the photosynthetic
with the exceptional reducing properties of MACC-38, suggests that this electron transport chain (PETC).
strain is particularly well-suited for application in BPV systems and may In photoheterotrophic cells, DCMU inhibited only 60–70 % of the
be able to support the function of microelectronic devices. In addition, light-dependent current (Fig. 3A). Acetate indirectly influences the
the outstanding exoelectrogenic properties of MACC-38 suggest that it redox state of the PETC by supplying stromal reductants that can reduce
could also be useful in the synthesis of value-added chemicals that photosystem I (PSI) (Kovács et al., 2000; Johnson and Alric, 2012).
require reducing power (Wang et al., 2021). These two points, however, Therefore, the incomplete inhibition of light-induced current produced
require more detailed investigation. in photoheterotrophically grown cells by DCMU may be attributed to
acetate-supported photosynthetic electron transport.
Since a significant portion of the electric current produced by MACC-
3.4. The impact of light intensity and metabolic conditions on the current
38 originates from its photosynthetic apparatus, varying the light in­
production of MACC-38
tensities during CA measurements could substantially impact the current
density output. Optimal output was observed at a light intensity of 150
To elucidate the primary physiological pathways that contribute to
µmol photons m− 2s− 1 (Fig. 3C), which was used for most CA measure­
the reduction of FeCN at the PM of MACC-38, the effects of the photo­
ments in this study. Light intensities exceeding this value resulted in a
synthetic inhibitor DCMU on MACC-38 cultures was examined under
progressive decrease in maximum current density (Fig. 3C), with the
different metabolic conditions.
light-induced current being completely abolished at 2000 µmol photons
Green microalgae, depending on the available carbon source for
m− 2s− 1 (Fig. 3C). These findings align with those of Thong et al. (2019),
assimilation, can exhibit photoheterotrophic growth (typically with
who reported that the maximum power density produced by suspension
acetate as a source of reduced carbon) or photoautotrophic growth in the
cultures of Chlorella sp. was achieved at 90 µmol photons m− 2s− 1 while
presence of CO2 (Johnson and Alric, 2012). When MACC-38 cells were
immobilized cells performed optimally at 150 µmol photons m− 2s− 1.
grown in acetate-containing (TAP) medium, approximately 43 % of the
The observed reduction in total current production at light in­
total current was light-induced (Fig. 3A). The application of DCMU, a
tensities above 150 µmol photons m− 2s− 1 may be due to photodamage,
photosynthetic inhibitor known to block photosynthetic electron
especially at intensities exceeding 1000 µmol photons m− 2s− 1.
transport at the QB binding site of photosystem II (Lavergne, 1982),

Fig. 3. Influence of metabolic state and light intensity on the electric current production by MACC-38. A) Effect of DCMU on the electric current production of
photoheterotrophically-grown MACC-38 strain. The graph shows mean values ± SE, with n = 6. B) Impact of DCMU on electric current production of
photoautotrophically-grown MACC-38. The graph shows mean values ± SE, with n = 6. C) Effect of light intensity on electric current production by MACC-38. The
graph shows mean values ± SE, with n = 6 for each light intensity. D) FV/FM values determined after chronoamperometric measurement at various light intensities.
The graph shows mean values ± SE, with n = 6 for each light intensity. Asterisks denote a statistically significant difference compared to the untreated variant (A, B).
Different letters signify statistically significant differences among light intensities (C, D). Statistically significant differences were determined using ANOVA with
Holm-Sidak post-hoc test at p < 0.05.

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N.Z. Petrova et al. Bioresource Technology 394 (2024) 130206

Supporting this hypothesis, FV/FM values of MACC-38 after current the first step towards characterization of the exoelectrogenic processes
production at 150 µmol photons m− 2s− 1 showed only insignificant of strain MACC-38. As expected, the ability of the four strains to reduce
changes compared to samples measured in darkness (Fig. 3D) while light FeCN paralleled their electric current productivity. MACC-38 stood out
intensities above 500 µmol photons m− 2s− 1 resulted in a significant with the highest FeCN reduction rate of approximately 1.9 mM h− 1
decrease in FV/FM (from approx. 0.6 to 0.4 at 2000 µmol photons (Fig. 4A). Strain 27.87 had an intermediate rate of about 0.9 mM
m− 2s− 1, Fig. 3D), indicating an inhibition of the photosynthetic appa­ h− 1(Fig. 4A). The lowest FeCN reduction rate was exhibited by strains
ratus with increasing measuring light intensity. 211–11g and 211-11c – approximately 0.4 mM h− 1 (Fig. 4A). Notably,
To prevent photodamage to the photosynthetic apparatus, various Gonzalez-Aravena et al. (2018) reported substantially lower FeCN
non-photochemical quenching (NPQ) mechanisms are activated to reduction activities in the range of a few µmol h− 1 for C. reinhardtii,
dissipate light energy (Erickson et al., 2015). Commonly observed NPQ Chlorella vulgaris and the cyanobacterial strain Synechococcus PCC7942.
mechanisms in green microalgae include energy (ΔpH)- dependent in­ The PM of plants contains numerous redox active groups (such as
crease in light energy dissipation (qE), changes in the pigment compo­ hemes, metals, flavins, etc.), suggesting a variety of electron transport
sition of the photosynthetic apparatus (qZ), reorganization of the processes occurring at the cell surface. However, only a small fraction of
pigment-protein supercomplexes in the photosynthetic membranes these processes have been identified and characterized (Luthje et al.,
(qT), or photodamage of the photosystems (qI). In addition, the effect of 2013). Given that FeCN cannot permeate cytoplasmic membranes, only
excessive light intensity can be mitigated by redirecting electrons in a subset of electron transfer reactions - those involving the donation of
PETC towards alternative electron transport routes (Erickson et al., electrons on the exterior side of the PM - are relevant to the experimental
2015). The activation of alternative electron pathways may have a direct conditions applied in the current work. In algae, the most extensively
effect on the overall redox state of the cells and therefore on their studied PM reactions that meet this criterion include: (i) the reduction of
exoelectrogenic activity. A detailed exploration of the NPQ mechanisms extracellular O2 to O•- 2 , which is necessary for molecular signaling and
and alternative electron transport processes in P. kessleri may provide for mounting an effective response to pathogen attack (Anderson et al.,
clarity on their effects on electric current production. 2011), and (ii) the reduction of trace elements (such as iron and copper),
which is a prerequisite for their uptake (Urzica et al., 2012; Merchant
et al., 2020).
3.5. Exploring the possible mechanisms of potassium ferricyanide
In C. reinhardtii, as observed in red algae and diatoms (Hervé et al.,
reduction in MACC-38
2006), the generation of extracellular O•- 2 is attributed to two homo­
logues of NADPH oxidase (NOX), namely RBO1 and RBO2. Previous
Elucidation of the unique molecular mechanisms underlying the
studies have indicated that the generation of extracellular O•- 2 is the
reduction of FeCN (and the electric current production) by MACC-38,
primary contributor to FeCN-mediated current production in
required the investigation and comparison of various PM redox activ­
C. reinhardtii (Anderson et al., 2016) and diatoms (Laohavisit et al.,
ities in the four P. kessleri strains.
2015). Furthermore, due to the ubiquitous presence of RBOs in virtually
Determination of the rate of FeCN reduction at moderate light was

Fig. 4. Exoelectrogenic properties of P. kessleri strains. A) Rate of FeCN reduction at moderate light intensity (150 µmol photons m− 2s− 1). The graph shows mean
values ± SE, with n = 3. B) Extracellular production of O•- 2 . The graph shows mean values ± SE, with n = 9. C) FRE activity expressed as the concentration of Fe(II)-
BPDS in the growth medium. The graph shows mean values ± SE, with n = 4. D) Cupric reductase activity as assessed by the concentration of Cu(I)-BCDS. The graph
shows mean values ± SE, with n = 4. E) Changes of extracellular pH upon addition of 5 mM FeCN to P. kessleri suspension cultures in the light. The graph shows mean
values ± SE, with n = 4. F) Effect of growth medium pH on the electric current production by MACC-38. The graph shows mean values ± SE, with n = 6. Different
letters signify statistically significant differences among P. kessleri strains (A, B, C, D) or pH treatments (F). Asterisks of different colors denote statistically significant
differences compared to the pH value of the respective suspension before the addition of FeCN (E). Statistically significant differences were determined using ANOVA
with Holm-Sidak post-hoc test at p < 0.05.

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N.Z. Petrova et al. Bioresource Technology 394 (2024) 130206

all organisms (Zhang et al., 2013), a brief screening of O•- 2 formation in the PM still await identification and characterization (Luthje et al.,
capacity has been proposed as an effective approach for predicting the 2013). Although their ability to reduce FeCN is a shared characteristic,
ability of microalgae to produce current in BPV (Anderson et al., 2016). the lack of information about their function, structure, and substrate
Based on this hypothesis, the extracellular O•- 2 accumulated by the four preferences precludes the performance of specific assays. However, it is
P. kessleri strains was quantified under conditions similar to those used plausible that the FeCN-reduction properties of MACC-38 are due to
during CA measurements in light. Interestingly, no correlation was previously unidentified PM redox protein(s).
found between the concentration of extracellular O•- 2 and the capacity One of the earliest observations regarding plant cells’ ability to
for current production. The O•- 2 produced by MACC-38 on a timescale reduce FeCN was that this reduction resulted in extracellular acidifica­
− 1
comparable to CA measurements was approximately 5 µM O•- 2 mg Chl tion (Belkoura et al., 1986; Marrè et al., 1988; Gonzalez-Aravena et al.,
(Fig. 4B), whereas the O•- 2 production by strains 27.87, 211–11g and 2018). This phenomenon was explained in terms of PM potential, with
− 1
211-11c was about ten-fold higher, in the range of 30–50 µM O•- 2 mg FeCN causing depolarization and H+ extrusion serving to restore the
Chl (Fig. 4B). charge balance across the membrane (Marrè et al., 1988). To detect a
Using sequence alignment, putative RBOs were identified in the similar effect in the four strains of P. kessleri, pH changes were monitored
MACC-38 strain by screening all coding sequences of the MACC-38 after adding FeCN to P. kessleri cultures suspended in unbuffered reac­
genome with C. reinhardtii RBO1 (Cre03.g188300) and RBO2 genes tion medium in the light.
(Cre03.g188400). Only low-identity matches were obtained (~40 % Contrary to the rest of the P. kessleri strains, adding FeCN to MACC-
identity across 90 % of the reference sequence) against RBO2 (see 38 caused a rapid and significant decrease in pH by about 1 unit in 20
Supplementary material) and no matches against RBO1. This is not min (Fig. 4E). In strains 27.87, 211–11g and 211-11c, a slight increase of
unexpected given the considerable phylogenetic distance between pH was observed (Fig. 4E). Notably, when MACC-38 was grown in
C. reinhardtii and P. kessleri. These findings suggest that while RBOs are buffered TAP medium with 5 mM FeCN, there was a decrease in pH by
present in the PM of MACC-38, they may operate at a lower rate than about 0.3 units compared to control cultures grown without FeCN (day
those in the other P. kessleri strains and C. reinhardtii (see Supplementary 4, data not shown).
material). To determine whether the observed acidification of the reaction
The reduction of Fe(III) represents another crucial exoelectrogenic medium benefits electric current production by MACC-38, CA curves
mechanism. The most prevalent form of iron in the environment, Fe(III), were recorded at various pH levels. At pH 5, the total current increased
cannot be absorbed by plant and algal cells without its reduction to Fe by about 25 %, and the light current increased by 80 % compared to
(II) at the PM. In C. reinhardtii, the enzyme responsible for this function values obtained at neutral pH (Fig. 4F). Alkaline medium had inhibitory
is ferric reductase (FRE, Allen et al., 2007; Urzica et al., 2012). Previ­ effect on current production. At pH 9, the total current was reduced by
ously, FRE activity was linked to FeCN reduction and in C. reinhardtii the approximately 35 % and the light current by about 25 % relative to the
ferric chelate-reductase and FeCN-reductase exhibited similar Km and values obtained at pH 7 (Fig. 4F). These results could indicate a pH
pH dependence, responded similarly to iron limitation and were thus optimum for the enzymes responsible for FeCN-mediated current pro­
argued to be represented by the same enzyme (Lynnes et al., 1998). duction - neutral to acidic pH seems to enhance FeCN reduction. On the
Therefore, the FRE activity of the four P. kessleri strains was evaluated other hand, the extracellular proton concentration is expected to affect
next. the polarization of the PM - a factor that has not been considered in
Unexpectedly, MACC-38 did not exhibit Fe(III) reduction activity FeCN-mediated electricity production but deserves more detailed
within the investigated timescale of 20 min (Fig. 4C). On the other hand, investigation in future studies.
strains 27.87 and 211-11c produced about 90–100 µM Fe(II)-BPDS mg− 1
Chl, and strain 211–11g showed the highest Fe(III) reduction activity, as 4. Conclusion
evidenced by the formation of about 300 µM Fe(II)-BPDS mg− 1 Chl
(Fig. 4C). Despite this, the MACC-38 genome contained homologues to One promising strategy for enhancing electricity production in bio­
the C. reinhardtii FRE1 gene (Cre04.g227400) with ~ 40 % identity photovoltaics is the utilization of algal strains with high exoelectrogenic
across 70 % of the reference sequence (see Supplementary material). capacity. In this study, a previously uncharacterized strain of the green
Additionally, homologues for Cre13.g586600, Cre14.g609900, and alga P. kessleri - MACC-38, was found to produce a significantly higher
Cre05.g241400 genes previously suggested to possess FRE-like activity electric current density than other P. kessleri strains and C. reinhardtii.
in C. reinhardtii (Urzica et al., 2012) were identified in MACC-38 as well Interestingly, MACC-38 revealed exoelectrogenic properties that differ
(see Supplementary material). from those typically observed in model green microalgae. MACC-38 is
Therefore, the markedly lower activities of RBO and FRE enzymes in particularly suitable for boosting the efficiency of long-term operating
P. kessleri MACC-38 show that the exoelectrogenic characteristics of this biophotovoltaic devices. Moreover, MACC-38 represents an excellent
strain significantly diverge from those commonly observed in green model for investigating the role of exoelectrogenesis on algal
algae. physiology.
In C. reinhardtii, cupric reductase, a PM protein, donates a single
electron to Cu(II), reducing it to Cu(I), which is subsequently trans­ Funding
ported into the cell (Hill et al., 1996). Although its activity has not been
evaluated in relation to electric current production, cupric reductase is a This work was supported by the National Research, Development,
potential contributor to the exoelectrogenic activity of microalgae. and Innovation Office (PD143438 and K132600, research grants to N. Z.
Among the P. kessleri strains, MACC-38 showed the highest Cu(II) P. and S.Z.T.), by the Lendület-Programme of the Hungarian Academy of
reduction activity, producing approximately 130 µM Cu(I)-BCDS mg− 1 Sciences (LP2020-5/2020 to G.M.), and by the European Union’s Ho­
Chl (Fig. 4D). Strains 27.87 and 211–11g exhibited substantially lower rizon 2020 Research and Innovation Programme under the Marie
Cu(I) production activity of 70–80 µM Cu(I)-BCDS mg− 1 Chl, while 211- Skłodowska-Curie grant agreement No 892632 (EnergUP project, to T.N.
11c had the lowest Cu(I)-BCDS formation of about 40 µM mg -1 Chl. T.).
(Fig. 4D). Therefore, the cupric reductase in MACC-38 is approximately
50–60 % more active than that of strains 27.87 and 211–11g, suggesting CRediT authorship contribution statement
that it may play a role in MACC-38′s exoelectrogenic activity. However,
other factors may also contribute to the exceptional FeCN-reduction Nia Z. Petrova: Conceptualization, Investigation, Formal analysis,
properties of MACC-38. Writing – original draft, Funding acquisition. Tünde N. Tóth: Investi­
As previously mentioned, a significant portion of the redox proteins gation, Funding acquisition. Prateek Shetty: Investigation, Formal

8
N.Z. Petrova et al. Bioresource Technology 394 (2024) 130206

analysis, Data curation. Gergely Maróti: Supervision, Funding acqui­ iron limited growth. RSC Adv. 8, 20263–20274. https://doi.org/10.1039/
C8RA00951A.
sition. Szilvia Z. Tóth: Writing – review & editing, Supervision, Funding
Herrero-Medina, Z., Wang, P., Lielpetere, A., Bashammakh, A.S., Alyoubi, A.O.,
acquisition, Conceptualization. Katakis, I., Conzuelo, F., Schuhmann, W., 2022. A biophotoelectrode based on
boronic acid-modified Chlorella vulgaris cells integrated within a redox polymer.
Bioelectrochemistry 146, 108128. https://doi.org/10.1016/j.
Declaration of competing interest
bioelechem.2022.108128.
Hervé, C., Tonon, T., Collén, J., Corre, E., Boyen, C., 2006. NADPH oxidases in
The authors declare that they have no known competing financial Eukaryotes: red algae provide new hints! Curr. Genet. 49, 190–204. https://doi.org/
interests or personal relationships that could have appeared to influence 10.1007/s00294-005-0044-z.
Hill, K.L., Hassett, R., Kosman, D., Merchant, S., 1996. Regulated copper uptake in
the work reported in this paper. Chlamydomonas reinhardtii in response to copper availability. Plant Physiol. 112,
697–704. https://doi.org/10.1104/pp.112.2.697.
Data availability Horváth, Á.N., Németh, L., Vörös, L., Stirk, W.A., Van Staden, J., Ördög, V., 2023.
Cataloguing microalgae and cyanobacteria strains from the Mosonmagyaróvár Algal
Culture Collection with in vitro antagonistic activity against phytopathogenic fungi
Raw sequencing data and assembled nuclear and chloroplast contigs and oomycetes. Phytoparasitica. https://doi.org/10.1007/s12600-023-01045-2.
of P. kessleri strains MACC-38, 27.87 and 211-11c have been submitted Huang, L.-F., Lin, J.-Y., Pan, K.-Y., Huang, C.-K., Chu, Y.-K., 2015. Overexpressing
ferredoxins in Chlamydomonas reinhardtii increase starch and oil yields and enhance
to NCBI, under accession numbers SRR26997537, SRR27029096 and electric power production in a photo microbial fuel cell. IJMS 16, 19308–19325.
SRR27027417, respectively, with BioProject IDs: PRJNA1046992 https://doi.org/10.3390/ijms160819308.
(MACC-38), PRJNA1047548 (27.87), PRJNA1047538 (211-11c). Jin, J.-J., Yu, W.-B., Yang, J.-B., Song, Y., dePamphilis, C.W., Yi, T.-S., Li, D.-Z., 2020.
GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle
P. kessleri strain MACC-38 is available upon request.
genomes. Genome Biol. 21, 241. https://doi.org/10.1186/s13059-020-02154-5.
Johnson, X., Alric, J., 2012. Interaction between starch breakdown, acetate assimilation,
Acknowledgments and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii. J. Biol. Chem.
287, 26445–26452. https://doi.org/10.1074/jbc.M112.370205.
Katz, A., Pick, U., 2001. Plasma membrane electron transport coupled to Na+ extrusion
The authors thank Dr. Kashif Shaikh Mohd for his help with genetic in the halotolerant alga Dunaliella. Biochimica et Biophysica Acta (BBA) -.
analyses and Bernadett Pap for the technical assistance in DNA samples Bioenergetics 1504, 423–431. https://doi.org/10.1016/S0005-2728(01)00157-8.
preparation. Gratitude is also expressed to Dr. Eugen Urzica (Kompe­ Kovács, L., Wiessner, W., Kis, M., Nagy, F., Mende, D., Demeter, S., 2000. Short- and
long-term redox regulation of photosynthetic light energy distribution and
tenznetz Darmerkrankungen, Münster, Germany) for the fruitful photosystem stoichiometry by acetate metabolism in the green alga, Chlamydobotrys
discussions. stellata. Photosynth. Res. 65, 231–247. https://doi.org/10.1023/A:1010650532693.
Laohavisit, A., Anderson, A., Bombelli, P., Jacobs, M., Howe, C.J., Davies, J.M., Smith, A.
G., 2015. Enhancing plasma membrane NADPH oxidase activity increases current
Appendix A. Supplementary data output by diatoms in biophotovoltaic devices. Algal Res. 12, 91–98. https://doi.org/
10.1016/j.algal.2015.08.009.
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org/10.1016/j.biortech.2023.130206. acceptor side of photosystem II. Biochimica et Biophysica Acta (BBA) -. Bioenergetics
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