Sakr et al.

Microbial Cell Factories (2023) 22:202 Microbial Cell Factories

https://doi.org/10.1186/s12934-023-02216-w

RESEARCH Open Access

Statistical optimization of waste

molasses‑based exopolysaccharides
and self‑sustainable bioelectricity production
for dual chamber microbial fuel cell by Bacillus
piscis
Ebtehag A. E. Sakr1*, Dena Z. Khater2, Zeinab M. H. Kheiralla1 and Kamel M. El‑khatib2

Abstract
Background The application of exopolysaccharide-producing bacteria (EPS) in dual chamber microbial fuel cells
(DCMFC) is critical which can minimize the chemical oxygen demand (COD) of molasses with bioelectricity produc‑
tion. Hence, our study aimed to evaluate the EPS production by the novel strain Bacillus piscis by using molasses
waste. Therefore, statistical modeling was used to optimize the EPS production. Its structure was characterized by UV,
FTIR, NMR, and monosaccharides compositions. Eventually, to highlight B. piscis’ adaptability in energy applications,
bioelectricity production by this organism was studied in the BCMFC fed by an optimized molasses medium.
Results B. piscis OK324045 characterized by 16S rRNA is a potent EPS-forming organism and yielded a 6.42-fold
increase upon supplementation of molasses (5%), ­MgSO4 (0.05%), and inoculum size (4%). The novel exopoly‑
saccharide produced by Bacillus sp. (EPS-BP5M) was confirmed by the structural analysis. The findings indicated
that the MFC’s maximum close circuit voltage (CCV) was 265 mV. The strain enhanced the performance of DCMFC
achieving maximum power density (PD) of 31.98 mW ­m−2, COD removal rate of 90.91%, and color removal of 27.68%.
Furthermore, cyclic voltammetry (CV) revealed that anodic biofilms may directly transfer electrons to anodes with‑
out the use of external redox mediators. Additionally, CV measurements made at various sweep scan rates to evaluate
the kinetic studies showed that the electron charge transfer was irreversible. The SEM images showed the biofilm
growth distributed over the electrode’s surface.
Conclusions This study offers a novel B. piscis strain for EPS-BP5M production, COD removal, decolorization, and elec‑
tricity generation of the optimized molasses medium in MFCs. The biosynthesis of EPS-BP5M by a Bacillus piscis strain
and its electrochemical activity has never been documented before. The approach adopted will provide significant
benefits to sugar industries by generating bioelectricity using molasses as fuel and providing a viable way to improve
molasses wastewater treatment.
Keywords Bacillus piscis, Optimized molasses-based media, EPS, COD removal, Decolorization, Electrochemical
activity

*Correspondence:
Ebtehag A. E. Sakr
Ebtehag.Abdelfattah@women.asu.edu.eg; ebtehagsakr@yahoo.com
Full list of author information is available at the end of the article

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Sakr et al. Microbial Cell Factories (2023) 22:202 Page 2 of 20

Introduction utilized as the substrates and the microorganisms present

The most serious issue now plaguing the globe is thought [9, 10].
to be climate change. The global depletion of fossil fuels Molasses is a syrupy by-product of the sugarcane
and its detrimental effects on the environment might be industry and has a thick, viscous, and dark brown color
seen as the fundamental obstacle to resolving the envi- [11]. Its use as a raw material for the generation of bio-
ronmental health issue. Therefore, the search for envi- fuels such as bioethanol, biodiesel, biogas, hydrogen,
ronmentally friendly technology is viewed as the most and methane as well as bio-electricity results in signifi-
promising method of producing sustainable energy [1, cant amounts of molasses wastewater [12]. Before dis-
2]. Among renewable energy sources like wind and solar charging it into the environment, a suitable wastewater
energy, microbial fuel cells (MFCs) are regarded as a treatment system is needed because dumping it into the
new sewage treatment method [3]. MFCs are employed water system could result in major pollution, including
to oxidize different organic substrates through bacte- foul odor and taste as well as effects on the city’s water
rial metabolism in order to produce bioelectricity and supply and aquatic inhabitants [13]. Some techniques,
eliminate pollutants at the same time [4, 5]. MFCs typi- including anaerobic treatment, adsorption, flocculation
cally come in one of two designs: a single chamber or precipitation, catalytic oxidation, and membrane separa-
two chambers divided by a proton exchange membrane tion, are the primary ways for treating molasses wastewa-
(PEM). The spotlight here is a dual-chamber MFC [6], as ter. These techniques share the traits of being expensive
illustrated in Fig. 1. and producing harmful byproducts. It is important to
The dual-chamber MFCs are appropriate for anaero- look at more suitable treatment options as we deal with
bic digestion, bioelectricity generation, wastewater the simultaneous challenges of energy shortages and
treatment, low energy consumption, low running costs, environmental pollution. Due to its poor fluidity, high
low sludge production, and clean techniques [7]. Cur- concentration of organic acids, high color, low pH, and
rently, the main factors impeding its advancement are significant corrosivity, molasses has not received much
poor power output, high cost, and inadequate waste- research, however some findings from earlier years have
water treatment [7]. Electricigens, the MFC’s structure, shown that it is an effective electron donor in MFCs [4,
the characteristics of the substrate, external and internal 8].
resistances, and the materials used for the electrodes are Molasses wastewater contains between 40 and 55%
the foremost issues that have an impact on performance. sucrose and has a pH between 5.5 and 6.5. It can be used
Simple molecules including sucrose, glucose, fructose, as a cheap substrate for MFCs because it contains the
ethanol, and acetate, as well as various wastewater from majority of the nutrients, such as sugar, protein, amino
domestic, industrial, and agricultural sources, can be acids, and vitamins, which improve the production of
used as substrates [8]. Several investigations have been biomass and EPS when used by microbes for the reduc-
done in the past using easily degradable sugars added tion of most molasses into small molecular sugars that
exogenously to the MFC to generate electricity. How- are easier for microorganisms to use [14]. According to
ever, the performance of the process varies in real-world the literature, total COD removal efficiencies attained
applications using wastewater depending on the wastes with MFC systems have ranged from 45.7 to 90.3%, and
the maximum power density (MPD) has been reported
to have ranged from 8.8 to 1410.2 mW ­m−2 [15, 16]. For
instance, a stacking MFC was created to handle molasses
wastewater, and its MPD was 115.5 mW ­m−2 [17].
Microbes linked to MFC produce a thin layer of micro-
bial growth called biofilm that adheres to the electrodes
[18]. The location where the biofilm develops controls
the bacteria’s ability to produce electrons and has a sub-
stantial impact on the disintegration of various organic
materials in MFC [18]. For the development of biofilms,
EPS and lipopolysaccharides are essential. Recent studies
have shown evidence that the EPS in electroactive biofilm
(EAB), which connects cells to cells over the electrode, is
electrically conductive [19]. The synthesis of EPS is often
regarded as one of the strategies employed by microor-
Fig. 1 A conceptual view of a dual-chamber MFC experimental ganisms to endure environmental stress [20]. Therefore,
system EPS producing bacteria was isolated from molasses.
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 3 of 20

Bacillus sp. has been proven to be a potent source of inverted sugar, ash content, and pH were 4.21%, 18.60%,
EPS production [21] and produces more varieties and 13.10%, and 5.6, respectively (Table 1).
larger amounts of EPS [22]. Rarely is molasses employed
in the production of polysaccharides [23]. Meanwhile, Isolation, selection, and genotypic identification
some polysaccharides such as pullulan, welan gum, and of the potent EPS producing isolate
glucan have all been produced from molasses [24–26]. Five colonies (1-Mol–5-Mol) isolated from molasses
For improved EPS production with the development of were chosen in light of the observation of mucous growth
a less expensive medium, the growth parameters were surrounding its colonies on agar plates. The isolate 5-Mol
optimized using Plackett–Burman design (PBD) and sta- was determined to have the largest EPS production
tistical design response surface methods (RSM). RSM (5.78 g ­L−1 of medium) than the other isolates, making it
is one of the effective statistical methods for planning a promising candidate for EPS production.
experiments, developing models, identifying the best The 5-Mol isolate was identified molecularly utilizing
combinations of variables to produce desired results, and the partially sequenced 16S rRNA genes and revealed to
assessing the relative importance of various influencing be a member of the genus Bacillus with a 99.88% identity
factors even in the face of complex interactions [27]. One to the species B. piscis with a query cover of 100%. A phy-
of the most widely utilized experimental models in the logenetic tree (Fig. 2) based on 16S rRNA gene sequences
RSM for maximizing the production of EPS from Bacil- was created to show the relative positions of this strain
lus licheniformis NS032 in a medium based on sugar beet (5-Mol) and other Bacillus species. The sequence was
molasses is the Box-Behnken Design (BBD) [28]. With additionally submitted into the GenBank database
this approach, a large number of factors can be optimized (NCBI) under accession number OK324045. The identi-
simultaneously, and a small number of experimental runs fied strain was designated as B. piscis strain 5-mol. B. pis-
can yield a lot of large quantitative information [29]. An cis has never before been isolated from molasses.
exciting area of research for industrial biotechnologists is
the optimization and characterization of innovative EPS- Statistical optimization of molasses based‑medium
BP5M synthesis from Bacillus sp. grown on inexpensive for EPS‑BP5M production by B. piscis
molasses using an ecologically friendly microbial method. Plackett–Burman design (PBD)
It is important to note that the research on EPS-BP5M Utilizing a cheap nutrient source like sugarcane molasses
and bioelectricity production from molasses optimiza- (industrial waste) is one of the cost-effective solutions for
tion via this strain has not yet been reported elsewhere. the growth of B. piscis. In this study, the PBD was used
This study was designed to screen the bacterial isolates in 12 runs to screen a total of 11 factors for their impact
from molasses for their ability to synthesize EPS-BP5M on the EPS-BP5M synthesis (Table 2). Bacterial EPS-
and identify the best candidate for bio-based polymer BP5M varied significantly from 12.12 to 27.50 g ­L−1. This
production. The EPS-BP5M yield was optimized by PBD
and RSM for the highest EPS-producing bacteria. Then,
the structural characterization of EPS-BP5M was done Table 1 The physicochemical parameters and EPS yield of
by Fourier-transform infrared (FTIR) analysis, proton bacterial isolates of sugarcane molasses
nuclear magnetic resonance (1H-NMR), elemental anal- Values
ysis, and High-Performance Liquid Chromatography
(HPLC). Also, the present study was focused on explor- Physicochemical parameters
ing the EPS producing Bacillus sp. for DCMFC electric- pH 5.60
ity production using optimized molasses as a growth Total sugar (TS), % 48.76
medium. Various parameters like OCV, CCV, cur- Total Fermentable sugars (TFS), % 39.98
rent density, polarization curve, CE, CV, and its kinetic Non-fermentable sugars (NFS), % 4.30
studies, and anodic biofilm were analyzed by SEM. The Residual sugar (RS), % 4.21
removals of COD and color presented in the molasses Inverted sugar, % 18.60
media were monitored. Ash 13.10
Brix 83.93
EPS yield (g/L) of bacterial molasses isolates
Results
1-Mol 2.94
Chemical analysis of sugarcane molasses
2-Mol 2.90
The amount of sugar was found to be fermentable
3-Mol 2.46
(39.98%) and un-fermentable (4.30%). 48.76% of the total
4-Mol 1.26
sugar can be regarded as a potential carbon source for
5-Mol 5.78
many microorganisms. The amounts of residual sugar,
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 4 of 20

Isolatee codes: 5-mol

Putatiive species: B. pisciss strain 5-mo
oll
Identity (%): 99.88
Accesssion No.: OK3240 045
Queryy Cover (%)): 100

Fig. 2 Phylogenetic relationships between 5-mol isolate and 16S rRNA gene sequences retrieved from the GenBank database

variation demonstrated the importance of optimization A Pareto chart (Fig. 3) illustrates how the t-value and
for achieving the highest yield of EPS-BP5M. The great- rankings are related. Depending on the significance level,
est EPS-BP5M yield (27.5 g ­L−1) was recorded in run No. the Pareto chart indicates the significance and magni-
1, whereas run No. 4 showed the lowest EPS-BP5M pro- tude of the factors that affect the EPS-BP5M produc-
duction (12.12 g ­L−1, Table 2). tion. Effects that exceed the t-value upper limit 2.776
The first-order polynomial (Eq. 1), which depicts the are considered significant. The three variables [molasses
association between each variable and the EPS-BP5M (A), ­MgSO4 (K), and inoculum size (H)] were discovered
yield, was constructed from the PBD data as follows: to have a substantial impact on the intended response of
  EPS-BP5M production based on the effects and P-values
Yield of EPS−BP5M gL−1 (Table 3).
= 13.52569 + 1.6575 ∗ Sugarcane molasses,
(X1) + 0.61583 ∗ Yeast extract, Box–Behnken design (BBD)
(X2) − 3.19667 ∗ NaCl, (X4) (1) The BBD of RSM was used to ascertain the ideal levels
+ 3.83611 ∗ MgSO4 , (X8) of the three chosen variables (molasses, M ­ gSO4, and
− 0.8675 ∗ MgCl, (X9) + 0.64042 inoculum size) based on the PBD analysis. Table 4 pre-
sents the design matrix and the associated responses. The
∗ Inoculum size, (X10)
findings of the experiment were examined using standard
− 0.70417 ∗ pH , (X11) ANOVA. The second-order polynomial equation (Eq. 2)
Molasses, ­MgSO4, MgCl, and inoculum size were was used to fit the BBD:
shown to significantly affect the production of EPS- Y (g L−1 ) =106.66 + 12.90X1
BP5M with P-values below the significance level in the
+ 201.38X2 + 27.37X3
statistical analysis using PBD (Table 3), while the remain-
ing components were determined to be insignificant with + 0.28X1X2 + 8.75E (2)
P-values above 0.05 for all of them. F-test was used to − 03X1X3 − 1.14X2X3
determine the significance of the fitting equation. The − 1.13X12 − 196.61X22 − 3.26X32
model was extremely significant (p = 0.0009 < 0.01) and
the ­R2 was 0.9013 and the adjusted R­ 2 was 0.9698, which where Y is the predicted EPS-BP5M production, X1, X2
indicated a good model fit. and X3 corresponded to molasses conc., ­MgSO4 ­conc.,
 Sakr et al. Microbial Cell Factories

Table 2 Optimization of EPS-BP5M production from B. piscis in molasses-based medium by PBD

Run F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F 11 Response ( EPS-BP5M
Yield)
(2023) 22:202

A: B: C: D: E: F: G: H: J: K: L: Actual Predicted
Molasses, (X1) Yeast Peptone NaCl, (X4) KH2PO4, (X5) K2HPO4, (X6) NaNO3, (X7) MgSO4, (X8) MgCl, (X9) Inoculum pH, (X11) value value
ex., (X2) ex., (X3) size, (X10)
% g/L g/L g/L g/L g/L g/L g/L g/L % pH g/L

1 8 2 2 0.5 0.3 1 5.0 0.8 0.5 6 8 27.5 27.26

2 2 2 2 0.5 0.1 1 2.5 0.2 0.5 2 6 14.05 13.86
3 8 4 2 0.5 0.1 2 2.5 0.8 2.5 2 8 23.88 24.20
4 2 2 2 1.0 0.1 2 5.0 0.2 2.5 6 8 12.12 11.68
5 8 4 2 1.0 0.3 2 2.5 0.2 0.5 6 6 24.98 26.00
6 8 2 4 1.0 0.3 1 2.5 0.2 2.5 2 8 18.63 19.07
7 2 4 4 1.0 0.1 1 2.5 0.8 0.5 6 8 17.22 16.95
8 2 4 2 1.0 0.3 1 5.0 0.8 2.5 2 6 14.3 14.06
9 2 2 4 0.5 0.3 2 2.5 0.8 2.5 6 6 16.05 16.99
10 8 2 4 1.0 0.1 2 5.0 0.8 0.5 2 6 25.03 24.51
11 8 4 4 0.5 0.1 1 5.0 0.2 2.5 6 6 26.89 25.87
12 2 4 4 0.5 0.3 2 5.0 0.2 0.5 2 8 13.5 13.69
F factor
Page 5 of 20
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 6 of 20

Table 3 Statistical analysis of the model by PBD for EPS-BP5M yield

Source Sum of squares df Mean square F value p value

Model 359.48 7 51.35 51.53 0.0009* Significant

A, (X1) 296.71 1 296.71 297.73 < 0.0001*
B, (X2) 4.55 1 4.55 4.57 0.0994
D, (X4) 7.66 1 7.66 7.69 0.0502
H, (X8) 15.89 1 15.89 15.95 0.0162*
J, (X9) 9.03 1 9.03 9.06 0.0395*
K, (X10) 19.69 1 19.69 19.75 0.0113*
L (X11) 5.95 1 5.95 5.97 0.0709
Residual 3.99 4 1
Cor total 363.47 11
R2 0.989
Adj ­R2 0.9698
Pred ­R2 0.9013
Adeq. precision 19.114
A: molasses; B: yeast ex.; D: NaCl; H: ­MgSO4; J: MgCl; K: Inoculum size; L: pH; DF: degrees of freedom
*
Significant at P<0.05. The rest of other factors were non-significant

Fig. 3 Pareto diagram of the fractional factorial design used to select the variables that affect the production of EPS-BP5M (a). Response surface 2D
counter plots showing interaction between sugar cane molasses vs. ­MgSO4 (b), sugar cane molasses vs. inoculum size (c), and ­MgSO4 vs. inoculum
size (d)
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 7 of 20

Table 4 Box–Behnken design for the production of EPS-BP5M

Run F1 F2 F3 Actual value Predicted value
A, molasses (X1) % B, ­MgSO4 (X2) g/L C, inoculum size (X3) % EPS-BP5M Yield (g/L)

1 8 0.5 2 18.27 17.71

2 8 0.2 4 12.91 13.90
3 5 0.5 4 37.11 37.11
4 5 0.2 2 4.10 3.67
5 5 0.5 4 37.11 37.11
6 8 0.8 4 14.86 15.39
7 8 0.5 6 21.83 20.87
8 2 0.5 6 9.45 10.01
9 5 0.8 6 7.28 7.71
10 5 0.5 4 37.11 37.11
11 2 0.5 2 6.10 7.06
12 5 0.5 4 37.11 37.11
13 2 0.2 4 4.17 3.64
14 2 0.8 4 5.10 4.12
15 5 0.8 2 5.99 6.02
16 5 0.5 4 37.11 37.11
17 5 0.2 6 8.12 8.09
F factor

Table 5 ANOVA of the different responses assessed for Box-Behnken design for EPS-BP5M
Source Sum of squares df Mean square F value p value

Model 2993.23 9 332.58 438.11 < 0.0001* Significant

A- (X1) 231.66 1 231.66 305.17 < 0.0001*
B- (X2) 1.93 1 1.93 2.54 0.154
C- (X3) 18.67 1 18.67 24.59 0.0016*
AB 0.26 1 0.26 0.34 0.5767
AC 0.011 1 0.011 0.015 0.9075
BC 1.86 1 1.86 2.45 0.1612
A^2 434.21 1 434.21 571.98 < 0.0001*
B^2 1318.37 1 1318.37 1736.68 < 0.0001*
^2
C 716.24 1 716.24 943.5 < 0.0001*
Residual 5.31 7 0.76
Lack of Fit 5.31 3 1.77
Pure Error 0 4 0
Cor Total 2998.54 16
R2 0.9982
Adj ­R2 0.9959
Pred ­R2 0.9716
Adeq. precision 50.083
A: molasses; B: ­MgSO4; C: Inoculum size; DF: degrees of freedom
*
Significant at P<0.05

and inoculum size, respectively. The proposed model’s be used to analyze the variation in the EPS-BP5M yield
F-value was 438.11 and its low P-value indicated that it because the model’s ­R2 value was 0.9716 and its adjusted
is highly significant according to the statistical analysis ­R2 value was 0.9959, demonstrating the model’s fitting
performed using ANOVA (Table 5). The model could of the tested model and confirming the high accuracy
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 8 of 20

and credibility of the test results [30]. To verify the fac- Structural characterization
tors’ significance, "Probe > F" was utilized, which denotes No absorption peaks at 260 or 280 nm were detected in
how strongly independent components interact with one the UV–Vis spectra of the EPS-BP5M (Fig. 4a), demon-
another. Because each "Probe > F" was less than 0.05, an strating the lack of proteins and nucleic acids [31].
ANOVA revealed that the model terms X1, X3, X ­ 12, ­X22, FTIR spectroscopy was used to examine the chemi-
2
and ­X3 were significant. cal composition of the EPS-BP5M produced by B. piscis
The interactions of molasses, inoculum size, and (Fig. 4b). The broad stretching peak of O–H stretching is
­MgSO4 for the production of EPS-BP5M are depicted evident at 3294 ­cm−1 [32]. At 2940 ­cm−1, C–H stretching
in a two-dimensional contour graph (Fig. 3). Because vibration has been found [32]. As a result of C–O stretch-
the contour plots were elliptical, significant interactions ing, the spectrum data also revealed a peak at 1656 ­cm−1
between the inoculum size and molasses and between [33]. Peak between 1000 and 1110 ­cm−1, which is indica-
the inoculum size and ­MgSO4 were also seen. tive of the presence of α-(1 → 4) Glup residue [34]. There
The regression equation’s predicted values were fre- was no peak for the β-configuration, which is expected to
quently in agreement with the experimental results, rise between 890 and 950 ­cm−1 [34]. The characteristic
demonstrating the model’s validity. According to the con- peaks of carbohydrates were a number of peaks between
tour plot and regression analysis, 5% molasses, 0.5 g ­L−1 1000 and 800 ­cm−1 [35]. Therefore, this molecule might
­MgSO4, and 4% inoculum size were the ideal conditions facilitate biofilm formation by B. piscis on our MFC.
to obtain the highest production of EPS-BP5M. The yield Most of the proton signals were found between 3.156
was found to be 37.11 g ­L−1 under these optimized con- and 4.591 ppm in the 1H-NMR spectrum of EPS-BP5M
ditions with a 6.42-fold enhancement as compared to the (Fig. 4c). Notably, two peaks (5.162 and 5.172 ppm) were
initial production medium (5.78 g ­L−1). These findings found in the anomeric region, indicating the existence of
suggest that EPS-BP5M from B. piscis can be produced anomeric protons. Hydrogen–deuterium oxide (HDO)
with a better yield during fermentation by using a waste is responsible for the peak at 4.70 ppm. Between 4.1 and
molasses-based medium as an alternative carbon source. 3.1 ppm, the frequency of HC–O (singly-oxygenated
hydrogen and carbon) chemical shifts was noted [36].

Fig. 4 UV (a), FTIR (b), NMR (c) spectra and monosaccharides composition (d) of EPS-BP5M from B. piscis
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 9 of 20

Table 6 Elementary composition of the EPS-BP5M and bacterial resistance of 10 kΩ over time following feeding with an
surface properties optimized molasses-based medium inoculated with B.
Parameters Values piscis. Our strain had three phases (log, stationary, and
decline phase) throughout the acclimatization period
Elemental composition of EPS-BP5M (%) when grown on molasses over three cycles. During the
C 28.56 log stage, each OCVs cycle started to climb linearly.
H 6.68 Throughout 200 h, the voltage outputs gradually stabi-
N 2.89 lized at maximum levels of 574, 599, and 624 mV, respec-
S 3.41 tively, with very minor changes for each cycle. After the
Bacterial surface properties OCVs dropped to extremely low values (about 70 mV)
Autoaggregation (%) due to the loss of fuel cell life, fresh molasses wastewater
3h 24.12 ± 3.25 was added. In such instances, the bacterial communities
6h 52.67 ± 1.62 in the functioning biological reactor already adjusted well
24 h 73.48 ± 1.79 to the environment of DCMFC and subsequently takes
Hydrophobicity (%) 31.98 ± 1.12 shorter periods to decrease the startup time for electric-
ity generation [37]. Moreover, Fig. 5a illustrated the influ-
ence of 10KΩ on the performance of DCMFC outputs
This suggests that EPS-BP5M may have a higher binding using molasses as an electron donor over three cycles of
capacity to the DCMFC electrode. Therefore, to establish operation. It could be observed that the CCVs had the
its ability to bind to electrodes, an experiment utilizing B. same previously pattern.
piscis producing EPS-BP5M is required.
The findings of the elemental analysis (Table 6) revealed Polarization characteristics DCMFC
higher concentrations of carbon and hydrogen, indicat- To ensure the accuracy of the measurement, several
ing that the majority of the components of EPS-BP5M external loads ranging from 650 kΩ to 100 Ω were
are sugars. applied when the OCV reached a plateau after feeding
with molasses to determine the representative steady-
Monosaccharide analysis state polarization properties and the accompanying
The EPS-BP5M was acid hydrolyzed, and its monosac- power density plots for DCMFC. The maximum power
charide composition was identified (Fig. 4d). The repeat- density ­(PDmax) was observed to be 31.98 mW ­m−2 when
ing units of this EPS-BP5M were observed by comparing the current density (CD) was 185.95 mA ­m−2 and the
the chromatographic results of the sample with the reten- resistance was 500 Ω at a corresponding voltage output
tion time of various monosaccharide standards. These of 172 mV (Fig. 5b). The calculated internal resistance
repeating units were glucose (73.6 mg/g) and fructose ­(Rin) was found to be approximately 463.4 Ω. Molasses
(10.0 mg/g). This result implied that strain 5-Mol syn- contained roughly 50% sucrose by weight, which was
thesized a heteropolysaccharide in an optimized molas- thought to be the primary substrate for the DCMFC. The
ses medium, with glucose serving as the major repeating equation for the oxidation of sucrose was determined
monomer in this polysaccharide chain. using a stoichiometric method similar to that used for the
oxidation of acetate [38].
The anodic reaction is then given as follows:
Cell surface characteristics
Bacillus sp. had hydrophobic cell surfaces and the cell B. piscis
C12 H22 O11 + 25H2 O −→ 12HCO− + −
3 + 60H + 48e
autoaggregation improved over time (31.98 ± 1.12%;
Table 6). The results clearly showed that EPS-BP5M (3)
alters the physicochemical characteristics of cell surfaces, whereas, the cathodic reaction as follows:
indicating that hydrophobic interaction may be crucial O2 + 4H + +4e− → 2H2 O (4)
for B. piscis cells’ ability to adhere to carbon-felt elec-
trode of our DCMFC. The polarization curve could be separated into three
zones. At the high cell voltage zone, the current increased
rapidly with the lowering of the cell voltage values, but
Utilization of optimized molasses‑based medium at the intermediate voltage zone, the rate of this increase
for electricity production using B. piscis strain was greatly diminished. At the low cell voltage zone, the
Voltage generation rate of the increased current became high again.
Figure 5a depicts the changes in open circuit voltages
(OCVs) and close circuit voltages (CCVs) at an external
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 10 of 20

Fig. 5 The performance of DCMFC inoculated with B. piscis and molasses wastewater as anode fuel, a Trend of both OPVs and CCVs output values
in the 800 h incubation period and b Power and polarization curve. c Color (ξcolor %) and COD removal efficiency of DCMFC after each cycle. d
Spectral scanning (visible band) of optimized molasses media before and after MFC treatment at 37 °C, following inoculation with B. piscis

COD removal efficiency monitoring the variations in COD during a period fol-
At the end of each cycle in the DCMFC system, the lowing the cell’s voltage reduction to less than 70 mV.
variation affinity of COD and its removal rate of the The CEs were 36.94, 33.22 and 21.23% at 12, 14 g ­L−1
molasses wastewater implemented as the anode sub- and 20 g ­L−1 COD, respectively. Additionally, the CE
strate medium were assessed (Fig. 5c) at an initial COD decreased as influent COD molasses concentration
concentration value was 22 g ­L−1. With a removal effi- increased. It could be confirmed the inverse relation-
ciency of 25 ± 3.2%, the COD averaged 16.5 g ­L−1 dur- ship between C­ E% and molasses concentration.
ing the OCV cycle. While with a removal efficiency of
59 ± 6.4%, the COD averaged 9 g ­L−1 during the CCV Color removal
cycle. Moreover, the maximum CCV at 265 mV was After DCMFC treatment, a decolorization of the molas-
obtained for 8 and 2 g ­L−1 while the minimum CCV ses was observed with a decolorization of 27.68 ± 1.52%
at 10 g ­L−1 was about 262 mV. As a result, the perfor- (Fig. 5c, d).
mance was due to the breakdown of molasses by elec-
troactive B. piscis, resulting in a lower COD value.
Cyclic voltammetry (CV)
The electrocatalytic activity of B. piscis biomass that had
Coulombic efficiencies been connected to the anodic electrode at various times
The oxidation of molasses over three cycles of opera- throughout the operation was confirmed by the CV anal-
tions causes a flow of electrons by mature B. piscis ysis. Thus, in order to determine the oxidation reduction
biofilm at the anode chamber, which is represented by activities and the mediators linked to the anodic cham-
the coulombic efficiencies (CEs%). It was calculated by ber, a sterilized molasses media inoculated with B. piscis
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 11 of 20

Fig. 6 The electrocatalytic activity of sterilized B. piscis in an anodic chamber for DCMFC using molasses as substrate, a CV before inoculation,
b, c recorded CVs at 20 mV ­s−1 for 2, 4, 5, 8, 11, 12 days, d the corresponding current density in correlation with day and e the trend of oxidized
and reduced peaks over the days
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 12 of 20

was incubated at various time intervals (0 h and 2, 4, 5, 8, relationship between peak current (Ip) and the square
11, and 12 days) at a scan rate of 20 mV ­s−1 (Fig. 6). root of scan rates (ʋ1/2) for oxidation and reduction
The CV displayed a smooth curve prior to inocula- peaks was used to calculate the Randles–Sevcik equa-
tion (0 h) with no redox peaks during the oxidation and tion (Fig. 7b, c). It was found that the Ip and the ʋ1/2 had
reduction process (Fig. 6a). While the B. piscis displayed a linear relationship. Moreover, the charge transfer at the
two reversible peaks, as shown in Fig. 6b, c, which point electrode happens more quickly than the active species
to the existence of redox peaks in the molasses-based diffuse from the bulk solution to the electrode surface.
media. Additionally, Fig. 6d, e depicts the trend of both Since, there were narrow, symmetrical faradaic peaks in
oxidized and reduced peaks over the days and the pro- in CV, the process might have been quick and reversible.
duced current densities. The voltage and current of oxi- The relationship between the peak potential (Ep) and
dized and reduced peaks were significantly shifted along scan rate (log ʋ) is seen in Fig. 7d, e. The slope values for
the operation time, which could be related to alterations the log Ip and log ʋ for the oxidized and reduced peaks
in the medium over time. Furthermore, redox peaks were nearly equal (0.35). When B. piscis was used as the
started to form at − 24 mV after 2 days of inoculation. source of the adsorption processes, the adsorbed nutri-
The oxidation and reduction peaks then started to rise at ents also appeared on the anodic electrode surface. These
− 26 mV after 4 days of incubation and ultimately reached findings demonstrated that the combination of diffusion
their peak after 5 days. The highest current densities and adsorption processes limited the kinetics, demon-
were recorded on days 5 and 8 of operation, measuring strating an irreversible electron charge transfer nature of
at 20.08 and 20.06 µA ­cm−2 with corresponding positive these peaks [43].
sweep potentials of 25 and 3.5 mV, respectively.
In addition, it was noted that the 11 day (− 125 and Biofilm characteristic
− 153 mV) and 12 day of operation (− 140 and 210 mV) After the DCMFC operation, the electrode’s surface was
had two oxidative waves. Reduction peaks were seen in examined using SEM. A dense biofilm can easily form in
the potential range between − 103 and − 720 mV during the gaps or holes between the electrode’s fibers (Fig. 8a).
the reverse scan. The released extracellular metabolites B. piscis was rod cells with a cell length ranging between
produced by B. piscis and the self-produced mediators 1.197 and 1.565 µm (Fig. 8a). The bacteria produced
that formed during the late exponential and stationary aggregates and gathered in groups, supporting the auto-
phases may be responsible for these waves [39]. After aggregation of the cells following treatment. This sug-
12 days, the peak current dropped to 6.6 µA ­cm−2 at gests that the aggregates are trapped in the fibrils, where
− 105 mV, presumably due to nutrient depletion, as no they eventually attach and grow. Bacterial adhesion and
medium refill was done [40–42]. These findings match colonization on the electrode surface would benefit from
with the cell proliferation displayed in Fig. 5a. The cell the mature EPS-based anodic biofilm.
growth peaked after 5 days of operation and entered the The findings of the EDX elemental mapping displayed
exponential phase. These results encourage further study in Fig. 8b demonstrated the presence of the elements C,
into whether B. piscis acts well as a biocatalyst for the N, O, Na, P, and S. Figure 8c presents the homogeneous
breakdown of molasses in MFCs, taking into account the distribution of basic elements. According to an elemen-
potential existence of EPS-BP5M which might accelerate tal analysis, the anodic biofilm contains a higher carbon
the electron transfer. content than any other element. The coexistence of EPS-
BP5M on the bioanode is confirmed by both EDX and
CV studies at different scanning rates elemental mapping.
On the 5th day, as shown in Fig. 7a, the CV was meas-
ured at various sweep scan rates of 5, 10, and 20 mV ­s−1 Discussion
to assess the kinetic studies on the inoculated DCMFC Microbial fuel cells (MFC’s) are gaining popularity owing
with B. piscis in the anodic chamber. The increase in scan to their eco-friendly and energy generation with biore-
rate was accompanied by an increase in redox peaks. mediation. Molasses are being explored as alternative
Additionally, the reduction peaks were modified towards low-cost nutrients to produce EPS-BP5M by B. piscis
a negative voltage value whereas the oxidized peaks were isolated from it (molasses). This study examined the effi-
shifted towards a more positive voltage value. Further- ciency of B. piscis in generating electricity and bioreme-
more, only the major peak of oxidation and reduction, diate the organic matter from molasses wastewater as
which was assumed to be the only simple electrode reac- anode substrate in DCMFC. To our knowledge, only two
tion, was studied. The direct electron transfer generation papers identified B. piscis strain. It was isolated from a
that was accomplished by the B. piscis may be the cause Dissostichus mawsoni muscle sample from the Antarctic
of the increase in peak value with scan rate. Also, the [44] and Indian salterns [45]. Different species of Bacillus
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 13 of 20

Fig.7 Kinetic study of B. piscis oxidation and reduction peaks at the 5th day of inoculation at the scan rates of 5, 10, 20 mV s.−1, a CV at different
scanning rates, b, c Ip at each redox vs square root of ʋ (Randles–Sevcik equation), d, e Ep vs log ʋ (plain diamonds) and log I vs log ʋ (black
triangles). Ep (peak potential); Ip (peak current); ʋ (scan rate)
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 14 of 20

Fig. 8 SEM images taken at different magnifications, bacterial cells (red arrow), bacterial attached to electrode fiber forming aggregates (blue circle)
(a); EDX analysis (b); and elemental mapping (c) of biofilm formed on carbon felt anode used in the DCMFC for molasses treatment and electrical
power generation

have been found in the wastes of the sugar industry [46, of polysaccharides [28]. Bacillus strains that produce EPS
47]. are more resilient to environmental stress ([54].
Any microbial strain can provide greater yields, but the Our findings supported previous findings indicating
key is to develop the production medium [48]. Several the carbon source had a significant impact on the forma-
factors affected the production of EPS including micro- tion of EPS [55–57]. The growth of microorganisms and
bial species, culture conditions, and nutritional types the production of EPS require a carbon source as a sub-
[49]. Molasses are useful as a growth medium because strate. Usually, EPS formation is encouraged by a high
they contain high levels of vitamins and minerals and concentration of a carbon source [58]. Due to its high
have a strong growth-stimulating impact [50]. It has been sucrose content, molasses was found to have a consider-
utilized as a substrate for the fermentation synthesis of able impact on the EPS-BP5M yield during the screening
biopolymers because of its various benefits, including of growth factors. Bacterial cells might use the additional
high sucrose and other nutrient levels, cheap cost, easy carbon supply to expand quickly and make more EPS at
availability, and simplicity of storage [51]. After optimiz- low concentrations of molasses, but at larger concen-
ing the process parameters, the maximum EPS-BP5M trations, catabolite suppression of oxidative pathways
production was 27.5 g ­L−1 which is higher than the bac- will occur, slowing down bacterial development [53,
terial EPS gained prior to the use of PBD (5.78 g ­L−1) by 59], because of an increase in osmotic pressure in the
4.76 times. RSM has been demonstrated to be a useful medium, which led to plasmolysis and cell death [53].
technique for evaluating the impact of factors on the pro- Our results indicated that the EPS-BP5M yield from B.
duction of EPS by B. licheniformis NS032 [28], Leuconos- piscis is higher than those of other microorganisms such
toc citreum B-2 [52], and Pantoea sp. BCCS 001 GH [31] as Bacillus subtilis (4.92 g ­L−1) [53], Bacillus licheniformis
using molasses as substrate. ANT 179 (16.35 g ­ L−1) [60], B. licheniformis mutant
−1
A high concentration of minerals and vitamins in strain (9.0 g ­L ) [54], and Pantoea sp. BCCS001GH
molasses, in addition to the sugars that are easily fer- (9.9 g ­L−1) [31].
mented, may be the cause of this increase in productiv- Auto-aggregation is associated with the development
ity [53]. The inhibiting effect of high quantities of mineral of biofilms [61], which would result if this also happened
components would be mitigated by the diluting of molas- within our DCMFC. EPS promotes cell aggregation
ses and nitrogen limitation can stimulate the formation through complicated interactions and also accelerates the
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 15 of 20

Table 7 Comparisons of current study performance and other similar studies inoculated with molasses wastewater as substrate
MFC construction Microorganisms used OCV (mV) CCV (mV) Power density COD % CE% References

DCMFC Bacillus piscis 624 265 185.95 mW ­m−2 90.9 36.9 Current study
H-shape DCMFC Meyerozyma guilliermondii NA 350 61.25 mW ­m−2 NA NA [1]
SCMFC Sludge NA 500 169.86 mW ­m−2 NA 13.3 [4]
DCMFC Sludge NA 7.5 31.37 mW ­m−2 35.5 NA [7]
U-shaped DCMFC Sewage 569 ~ 351 88.9 mW ­m−2 NA 81 [8]
DCMFC Brevibacillus borstelensis 990 453 188.5 mW ­m−2 81.7 59.8 [12]
Baffled stacking MFC Sludge 690 115.5 mW ­m−2 70 1 [17]
DCMFC Yeast NA 372 813.19 mW ­m−2 97 NA [62]
SCMFC Candida boidinii 953 5.45W ­cm−2 NA NA [64]
DCMFC Pseudomonas Sp NA 670 660.82 mW ­m−2 NA NA [72]
DCMFC Sludge NA 392 14.9 mW ­m−2 31.8 6.2 [73]
SCMFC single chamber MFC, DCMFC dual chamber MFC, NA not applied

development of biofilms and granulation [62]. In the field methanogenesis, which take place in the anode chamber
of wastewater treatment, the ability of microbial cells to through the breakdown of a portion of molasses, could
auto-aggregate is intriguing as this may encourage the be the cause of the reduction of CE in the MFC [68].
production of bioaggregates and control the efficacy of Molasses wastewater has a dark brown color due to
treatment [63, 64]. the presence of amounts of melanoidins, caramel, and
Bacillus sp. is simple to grow and produces EPS with a phenolic pigments present. Melanoidins are the hardest
high potential for bioremediation [59]. B. piscis was well to get rid of because it has the largest content and most
adapted on the surface of the carbon felt electrode, gen- complex structure of all of them [69]. After DCMFC
erating a mature electrogenic biofilm, according to stable treatment, a decolorization rate can break down com-
voltage outputs [3, 17]. The power density for the pre- plex compounds like melanoidoin, producing colorless
sent study was found to be comparable with many of the intermediate metabolites [70]. The rates of decoloriza-
reported literature values as shown in Table 7. The ­PDmax tion in the current study are higher than those previously
values were higher than those examined by Fan et al. reported by Mohanakrishna et al. [71], who reported
(2016). They utilized a DCMFC fueled with simulated 22.92% of decolorization following MFC treatment of
molasses wastewater as the anode substrate and showed distillery effluent inoculated with mixed consortia.
­PDmax generation of 31.37 mW m ­ −2 with a matching volt- B. piscis’ electrocatalytic activity is supported to regu-
age output of about 7.5 mV [7]. Additionally, Lee et al. late subsequent oxidation processes and intermediate
(2016) reported in another investigation that the DCM- breakdown [12]. The linear relationship of Ip and the ʋ1/2
FC’s highest CD was 80 mA ­m−2 and PD was 17 mW ­m−2 indicated the performance was subjected to a diffusion-
[65]. In contrast, our finding was lower than the research controlled process [72]. Excellent and efficient adsorption
conducted by Syafitri et al. (2018). They reported that the occurs through the electrode holes because it enhances
DCMFC produced the maximum voltage (0.372 V) and the contact between pollutants and exoelectrogens [73].
­PDmax (813.191 mW ­m−2) when sediment was mixed with Bacterial adhesion and colonization on the electrode sur-
molasses [66]. The discrepancy in power density values face would benefit from the mature EPS-based anodic
revealed in various publications may be due to variations biofilm. Consequently, it might be said that the bacteria
in substrates, microbial species, MFC configuration, elec- were metabolically active and increased power output
trode materials, and PEMs. Our novel B. piscis exhibits [74].
good performance on electricity generation using molas-
ses wastewater as anode fuel. Conclusions
As the COD concentrations decreased, the overall In the present study, a novel EPS-producing bacteria, B.
COD removal efficiency increased yields by 54.55, 63.64, piscis, was isolated and identified from molasses waste.
and 90.91%, respectively. These findings suggested that The efficient EPS-BP5M production in an optimized
the B. piscis activity is more potent and may be employed medium with inexpensive sugarcane molasses substrate
for COD elimination and voltage generation from was also carried out which resulted in higher EPS-BP5M
molasses [3, 67]. The fermentation mechanisms, bio- production. To stick to the carbon felt electrode of the
mass formation, and consumption of coulombs during DCMFC, the bacterium most likely generates EPS-BP5M.
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 16 of 20

This work offered new insights on the use of bacteria that NCBI database. The bacterial sequence was submitted
produce EPS using molasses. EPS-BP5M was character- to the GenBank database and the accession number was
ized using FT-IR, NMR, and HPLC. B. piscis could be obtained. The isolated bacteria were then aligned, and
used as an anodic biocatalyst for the treatment of sugar MEGA software version 11 was used to create phyloge-
industry molasses used as a substrate in a DCMFC and netic trees using the neighbor joining method.
simultaneous energy recovery. Our findings show an
effective and eco-friendly approach (DCMFC) that uses Molasses‑based media optimization
molasses, a sustainable agricultural by-product, to solve Pretreatment of molasses
the problem of eliminating organic matter and produc- Molasses used as a carbon source were mixed with dis-
ing bioelectricity.. Further work will be done on molasses tilled water containing 2% sodium dihydrogen phosphate
wastewater treatment. (1:1). The solution was autoclaved at 121 °C for 10 min
before being allowed to cool for 24 h. Clarified molasses
Material and methods was the only source of carbon used [76].
Collection and analysis of sugarcane molasses
The sugar company for integrated industries, located Fermentation media
in Hawamdiya, Giza, Egypt, kindly donated the sugar- During the fermentation procedure, a 250 mL flask was
cane molasses. The collected material was carried to filled with 100 mL of medium. The production medi-
the lab with care, stored immediately at 4 °C, and then um’s composition varied depending on the experimental
brought to room temperature before usage. According to approach outlined below. Depending on the experimental
the Association of Official Analytical Chemists (AOAC) design, the best isolate (50 × ­106 CFU/mL) was employed
guidelines, the chemical composition was examined [75]. to inoculate the sterilized medium. The cultivation pro-
cess took place at 37 °C for 48 h. In order to calculate the
Isolation of EPS producing bacteria yield of EPS-BP5M (g ­L−1) at the end of the fermentation,
First, Erlenmeyer flasks holding 25 mL of liquid nutrient samples from the liquid culture were taken.
broth (NB) medium were inoculated with 1.0 mL of sug-
arcane molasses and incubated at 37 °C for 48 h. Then, Experimental designs
1.0 mL of that culture was transported to petri dishes PBD was used to organize 11 independent variables at
with nutrient agar medium amended with 1% sucrose two levels with 12 runs to rank the impact of various fac-
and incubated for 48 h at 37 °C. Striking was used to sep- tors on EPS-BP5M yield (Table 2). The tested variables
arate the mucoid colonies into pure cultures, which were were molasses, yeast extract, peptone extract, NaCl,
then frozen at − 20 °C in NB medium with 25% glycerol. ­KH2PO4, ­K2HPO4, ­NaNO3, ­MgSO4, ­MgCl2, inoculum
size, and pH.
EPS production, extraction, and purification According to the PBD experiment, molasses, ­MgSO4,
Bacterial isolates were screened for their ability to pro- and inoculum size were further optimized using the
duce EPS by inoculation of NB medium supplemented RSM. As shown in Table 4, variables were performed at
with 1% sucrose. Then, the inoculated flasks were incu- three-level (low, middle, and high) trials. Design-Expert
bated at 37 °C for 48 h. The fermentation broth was cen- software created a 17-run experimental design scheme
trifuged at 6000 rpm for 10 min, and the supernatant was in accordance with the coding design, utilizing the yield
then mixed with Savage reagent (chloroform: n-butanol, of EPS-BP5M (Y) as the response value at the end of
2:1 v/v). The organic layer was recovered, combined at fermentation.
a ratio of 1:3 (v/v) with cold absolute ethanol, and then Design-Expert 7.0 software was used to output the
left to stand at 4 °C overnight. The precipitate was col- results of variance analysis of the PBD experiment and
lected and mixed with ultrapure water to create a crude Box–Behnken experiment and carry out regression anal-
EPS solution. This solution underwent a 72-h dialyze in ysis of the results of the Box–Behnken experiment to
deionized water to produce an EPS. For the following tri- simulate the prediction equation.
als, the isolate that produced the highest yield of EPS was
chosen. Structural characterization
Ultraviolet–visible (UV–Vis) spectroscopy of the sample
Molecular analysis of the selected EPS producing isolate was evaluated using a Shimadzu UV-1800 UV spectropho-
Using 16S rRNA gene sequencing, the chosen bacteria tometer with a wavelength range of 200–600 nm. Using an
were identified. Using universal 16S rDNA primers, the FTIR (Bruker Alpha 11), the EPS-BP5M sample’s functional
extracted and purified DNA was amplified. The ampli- group content was initially validated. One mg of the sample
fied product was sequenced and deposited into the was combined directly with KBr and then quantified using
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 17 of 20

a spectrum. EPS-BP5M’s. 1H-NMR spectrum was obtained Synergistic interactions of DCMFC and microbially
utilizing a BRUKER 400 MHz spectrometer at 25 °C. Fol- induced removal of color and COD of molasses waste
lowing that, the material was dissolved in ­D2O at a 50 mg/ as the substrate
mL concentration. Parts per million (ppm) were used to DCMFC construction and operation
describe chemical changes. The EURO EA Elemental Ana- The experimental system was mainly made up of a dual-
lyzer was used to conduct an elemental analysis on the car- chamber MFC reactor that was assembled by joining two
bon, hydrogen, nitrogen, and sulphur weight percentages cylindrical plexiglass chambers that served as the anodic
of EPS-BP5M. Approximately 1.0 mg of the material was and cathodic chambers. These chambers were 6 cm long,
burned. Gas chromatography is used to quantify the com- 4.6 cm in diameter, and had a total working volume of
bustion byproducts (C, H, N, and S), and the ratio of the 100 mL. Proton exchange membrane (PEM, Nafion 117,
original sample’s constituents is then calculated. Dupont Co.), which assures the transmission of only
hydrogen ions and no other ions, was used as the sepa-
Monosaccharide composition rator between the anode and cathode. The anode com-
The monosaccharide compositions were analyzed by prised an unmodified sheet of three-dimensional carbon
HPLC using a Shim-pack SCR-101N column with a mobile felt joined to the top of an externally connected anode
phase of ultrapure water. A paste was made by carefully port with affective dimensions of 2.5 × 2.5 × 0.6 cm and
adding 0.5 ml of ice-cold 80% H ­ 2SO4 to the EPS-BP5M a projected surface area of 18.50 ­cm2. The cathodic elec-
sample. After that, the paste was carefully mixed for 15 h trode was made of non-waterproof gas diffusion carbon
at room temperature. The paste was then diluted with a cloth with a microporous sheet (6 × 6 cm each; surface
solution of ice and distilled water (up to 6.5 mL) until the area equivalent to 16.63 ­cm2). A stainless steel wire was
sulfuric acid strength reached 2N. The solution was fur- used to link the electrodes in bioreactors so that electrons
ther hydrolyzed for 6 h in a sealed tube over a boiling water could be transferred. A 50 mM phosphate buffer solution
bath. The hydrolyzate that was produced was neutralized was employed in the cathode chamber, which was left
by ­BaCO3 before being filtered and thoroughly rinsed with open to the atmosphere for ventilation of ­O2 as an elec-
water. After that, a cation exchange resin (Amberlite IR-120 tron acceptor, while the anode chamber was completely
(H +)) was used to treat the filtrate and washings. A flow sealed with epoxy sealant to maintain an anaerobic envi-
rate of 0.7 ml/min was used to analyze the monosaccharide ronment [42, 79]. The anode substrate was obtained in
content. After integrating the relevant areas and comparing fed-batch mode using the molasses wastewater (g ­L−1)
the results to standard curves made from glucose, fructose, with 50 mL as the carbon source, 2.0 g yeast extract, 2.0 g
sucrose, and arabinose (Sigma), each carbohydrate concen- peptone, 0.5 g NaCl, 0.1 g ­KH2PO4, 1.0 g K ­ 2HPO4, 2.5 g
tration was calculated. ­NaNO3, 0.5 g M ­ gSO4, 0.5 g M
­ gCl2, and 4% inoculum size.
The electrolyte was adjusted to pH 7 with NaOH. B. pis-
Bacterial surface properties cis that had first been activated overnight in NB medium
Three subcultures of the isolate were performed in NB was inoculated into DCMFC to aid in the breakdown
medium at 37 °C. After centrifuging the active cultures for of molasses. The strain was cultivated in 100 mL of NB
5 min at 6000 rpm, they were washed with sterile saline medium for 24 h at 37 °C, followed by a 10-min centrifu-
solution. The washed pellets were combined with sterilized gation at 6000 rpm at 4 °C. Phosphate-buffered saline
phosphate-buffered saline (PBS) buffer for autoaggrega- (PBS) was used to wash the cell pellets before they were
tion [77] or saline solution for hydrophobicity [78], and the adjusted to 50 × ­106 CFU/mL. After the sugarcane molas-
­OD600 nm was adjusted ­(A0, ­H0). For the auto-aggregation ses was consumed and the decline phase, fresh B. piscis
assay, the bacterial culture (8 mL) was incubated at 37 °C, and molasses were added to the anodic. The experiments
and the auto-aggregation values were recorded at 3, 6, and were run at room temperature.
24 h ­(At). It was determined using the equation (%) = 100
* [1 − ­(At/A0)]. To determine the hydrophobicity of the cell DCMFC analysis and calculations
surface, hexadecane was employed as a solvent. The cell The voltage of the DCMFC was recorded every 5 min
suspension was mixed with hexadecane, and the process using a data acquisition system (Lab jack U6-PRO) con-
was vortexed for 1.0 min. A ­ t 600 nm (­H1), the optical den- nected to a laptop, and Ohm’s Law (V = IR) was used to
sity of the aqueous phase was determined after 15 min of determine the current value when the external resistance
separation. The equation affinity (%) = 100 * [1 − ­(H1/H0)] was set to 10 kΩ. Open circuit voltage (OCV), which
was then used to compute the percentage of cells that were was obtained in the steady state when the circuit was
transported to the hexadecane phase. opened. Polarization and power curves were generated
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 18 of 20

by manually stepping down the external resistance from Biofilm characterization

650,000 to 100 Ω. The current and power was normalized Using scanning electron microscopy (SEM Quanta FEG
to the anode surface area to calculate the current density 250 with field emission gun, FEI Company, Netherlands),
(mA ­m−2) and power density (mW ­m−2). Internal resist- the surface morphologies and microstructures of the
ance is determined by the slope of the linear region of anodic biofilm were studied. The biofilm was performed
the current–voltage graph. According to APHA standard by fixing in 0.1 M phosphate buffer (pH 7.0) contain-
procedures, COD concentrations in influent and efflu- ing 2.5% (v/v) glutaraldehyde for an overnight period
ent were evaluated. Organic concentrations were esti- [81]. The increasing gradients of ethanol (25, 50, 70, and
mated as COD removal efficiency (COD R%), which was 100%) were used to induce dehydration. The biofilm was
calculated using the following equation: COD R % = 100 allowed to dry at room temperature before being sputter-
* [(CODinitial − ­CODfinal)/CODinitial], where C
­ ODinitial is coated with gold and subjected to analysis at a voltage
the COD concentration in the influent (mg COD/L), and 20 kV. Additionally, energy-dispersive X-ray spectros-
­CODfinal is the COD concentration in the final effluent copy (EDX) was used to determine the element content
at the end of DCMFC batch cycles (mg COD/L) [4]. The and elemental distribution maps.
coulombic efficiency (­CE) expresses the charge efficiency
in which electrons were being transferred in the DCMFC. Statistical analysis
It was calculated by dividing the generated current to the The statistical analysis was done using the Microsoft
theoretical current as CE = [M∫I.dt / nFvΔCOD] * 100, Excel 2016 version. Data analysis dealt with all variables,
where I is current output (A), v is the working volume (L) computations, means, and standard deviations.
in the anode, n is the number of electrons exchanged per
­ 2 = 4, F is Faraday’s constant (96,485 A s ­mol−1),
mole of O Acknowledgements
This research article was financially supported by Ain Shams University, Egypt.
ΔCOD is change in chemical oxygen demand (g ­L−1), M
the molar weight of oxygen [8]. Author contributions
ES designed the project. ES and DK carried out experiments and drafted the
manuscript. ES, DK, ZK, and KE designed the methodology and revised the
Color removal manuscript. ZK supervised the project. All authors read and approved the final
After DCMFC treatment, molasses samples were diluted manuscript.
1:10 in triplicate, the pH was set to 7.0, and they were Funding
then subjected to a spectral scan by the UV-1800 spec- Open access funding provided by The Science, Technology & Innovation
trophotometer from wavelengths between 350 and Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). The study was financially supported by Ain Shams University, Egypt.
650 nm to assess the decolorization. The formula used to
determine color removal efficiency (ξcolor %) at 475 nm Data availability
(the wavelength at which melanoidins absorb best) is All data generated or analyzed during this study are included in this article.
ξcolor % = 100 * [1 − ­(Ai/Af)], where ­Ai and ­Af refer to ini-
tial and final absorbance, respectively. The samples were Declarations
taken after each cycle to measure the color [70]. Ethics approval and consent to participate
This study was approved by the Ethics Committee of Women Faculty for Arts,
The electrochemical measurements Science, and Education, Ain Shams University, Cairo, Egypt.

Cyclic voltammetry (CV) of sterilized DCMFC that inoc- Consent for publication
ulated with a single culture of B. piscis (cell suspension Approved by all named authors.
of 50 × ­106 CFU ­mL−1 at logarithmic phase) was achieved
Competing interests
by applying various scan rates at 5, 10, and 20 m mV ­s−1 The authors declare that they have no competing interests.
at different testing periods from 24 to 288 h after cell
operation. The CVs were collected using the Voltamaster Author details
1
Botany Department, Faculty of Women for Arts, Science and Education, Ain
6 potentiostat (PST006) in the potential range of − 0.8 Shams University, Cairo, Egypt. 2 Chemical Engineering and Pilot Plant Depart‑
to 0.8 V vs. Ag/AgCl. The anode, cathode, and Ag/AgCl ment, National Research Centre (NRC), El Buhouth St., Cairo 12622, Dokki,
(Metrohm) were implemented as working, counter, Egypt.

and reference electrodes, respectively. The study of the Received: 27 July 2023 Accepted: 26 September 2023
reversibility of the electron transfer was depicted by the
peak potential dependence on scan rate and linearity of
the Randles–Sevcik equation (linear plot of peak current
dependence on the square root of scan rate) [80].
Sakr et al. Microbial Cell Factories (2023) 22:202 Page 19 of 20

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