Science of the Total Environment 547 (2016) 197–205

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Conversion of orange peel waste biomass to bioelectricity using a

mediator-less microbial fuel cell
Waheed Miran, Mohsin Nawaz, Jiseon Jang, Dae Sung Lee ⁎
Department of Environmental Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea

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

• Orange peel waste (OPW) was explored

for bioelectricity generation in MFCs.
• A maximum stable voltage of 0.59
± 0.02 V was generated using OPW.
• Microbial communities were analyzed
by high throughput 16S rRNA pyrose-
quencing.
• Enterococcus, Paludibacter, and Pseudo-
monas were dominant genera in anode
biofilm.
• Exoelectrogen and fermentative bacte-
ria played significant role in MFC per-
formance.

a r t i c l e i n f o a b s t r a c t

Article history: Microorganisms have the potential to become a game-changer in sustainable energy production in the coming
Received 12 August 2015 generations. Microbial fuel cells (MFCs) as an alternative renewable technology can capture bioenergy (electric-
Received in revised form 29 December 2015 ity) from carbon-based sources by utilizing microorganisms as biocatalysts. This study demonstrated that MFC
Accepted 3 January 2016
technology can be explored for bioelectricity production from orange peel waste (OPW), an agricultural
Available online xxxx
byproduct and an organic substrate, without any chemical pretreatment or the addition of extra mediators. A
Editor: Simon Pollard maximum voltage generation of 0.59 ± 0.02 V (at 500 Ω) was achieved in a dual chamber MFC during stable volt-
age generation stages. The maximum power density and current density obtained were 358.8 ± 15.6 mW/m2
Keywords: and 847 ± 18.4 mA/m2, respectively. Key components of OPW, namely pectin and cellulose, were also tested
Bioconversion in their pure form, with pectin giving a stable current, while no significant current generation was achieved
Waste treatment using cellulose alone as the substrate, thus demonstrating the absence of cellulose-degrading bacteria. Maximum
Enzyme activity pectinase and polygalacturonase enzyme activities of 18.55 U/g and 9.04 U/g (per gram of substrate), respectively
Microbial growth were achieved during orange peel degradation in MFCs. Bacterial identification using 16S rRNA analysis of the ini-
Microbial fuel cell
tial inoculum fed to the MFC, the biofilm attached to the anode, and the anode suspension, showed significant
Microbial community
diversity in community composition. A well-known exoelectrogen, Pseudomonas, was present among the pre-
dominant genera in the anode biofilm.
© 2016 Elsevier B.V. All rights reserved.

⁎ Corresponding author.
E-mail address: daesung@knu.ac.kr (D.S. Lee).

http://dx.doi.org/10.1016/j.scitotenv.2016.01.004
0048-9697/© 2016 Elsevier B.V. All rights reserved.
198 W. Miran et al. / Science of the Total Environment 547 (2016) 197–205
1. Introduction
The future of energy sustainability and supply is likely to rely on re-
newable energy sources. The production of electricity or biofuels using
innovative technologies and renewable sources is a global priority in
terms of energy strategies (Resch et al., 2008). A microbial fuel cell
(MFC) is an emerging renewable technology, which is designed to ex-
ploit the degradation of biological substrates for the production of sus-
tainable bioenergy in the presence of active microorganisms (Kan
et al., 2011; Walter et al., 2015). Moreover, this novel electrochemical
device presents a more practical alternative to existing technologies
for energy recovery as it directly transforms organic waste to electricity
(Kelly and He, 2014).
Electricity generation in MFCs is favored by numerous substrates,
extending from easily biodegradable pure substrates such as glucose
(Rabaey et al., 2003), ethanol (Kim et al., 2007), and acetate (Logan
et al., 2007), to complex substances such as starch (Herrero-
Hernandez et al., 2013), chitin (Rezaei et al., 2009b), and cellulose
(Rismani-yazdi et al., 2007). However, utilization of these substrates
in MFCs is not a cost effective option. Biomass being a carbon neutral
has been attracted as one of the most promising future resources of
electricity generation for addressing the rapidly growing energy needs
(Mao et al., 2015). Therefore, cheap biomass sources, such as raw corn
stover (Wang et al., 2009), rice (Hassan et al., 2014) and wheat
(Zhang et al., 2009) straw hydrolysate, and algae powders (Velasquez-
Orta et al., 2009) have also been tested as substrates in MFCs. Many
other forms of waste biomass exist, each containing large amounts of
energy and can be a better source of power generation in MFCs. These
remain generally unexploited and yet to be tested in MFCs for microbial
degradation and ultimately bioelectricity generation.
Oranges are citrus fruits that are consumed worldwide in large mag-
nitudes in their natural, peeled, and juiced forms (Rezzadori et al.,
2012). The statistical database of the Food and Agriculture Organization
of the United Nations (FAOSTAT) shows that worldwide orange produc-
tion was approximately 68.2 million tons in 2012, representing nearly
52% of the total citrus fruit production (http://faostat3.fao.org/home/
E). During orange juice production, approximately 50–60% of the proc-
essed fruit weight is converted to peel waste, comprised mainly of peel,
seeds, and membrane residues (Garcia-Castello et al., 2011; Wilkins
et al., 2007). This large volume of waste is either deposited on soil
near the production site for use as an animal feed raw material after dry-
ing, or is incinerated. Such a method of waste handling results in waste-
water pollution in terms of chemical and biological oxygen values,
which can negatively affect the soil, ground, and superficial waters
(Martín et al., 2010). A number of promising proposals for use of this
waste have been described, including the production of fertilizers, es-
sential oils, pectin, industrial enzymes, single cell proteins, pollutant ab-
sorbents, and paper pulp supplements. In addition, in terms of useful
bioenergy recovery, ethanol production (Choi et al., 2013; Oberoi
et al., 2010) and anaerobic digestion to produce methane gas (Koppar
and Pullammanappallil, 2013) are believed to be viable treatment
methods, and benefit from abundant orange peel waste (OPW). An ex-
cellent alternative approach to ethanol and methane gas production
from OPW is bioelectricity production using an MFC, thus avoiding en-
vironmental pollution, and reducing operating costs.
This study explores OPW as an economical and feasible substrate for
bioelectricity generation, in parallel with a reduction in chemical oxy-
gen demand, thus resulting in environmentally friendly degradation of
the OPW. To the best of our knowledge, electricity production in MFCs
using OPW has not yet been reported. Voltage generation, power den-
sity, polarization curves, coulombic efficiencies, and organic matter re-
moval were monitored to investigate the performance of this system.
In addition, microbial community structure on the anode, in suspension,
and in the initial inoculum of the MFC is analyzed using high throughput
pyrosequencing.
2. Materials and methods
2.1. Orange peel waste and anaerobic sludge
Fresh oranges (Citrus sinensis) were purchased from a local market
in Daegu, South Korea. Oranges were manually peeled and processed
for use in the MFC. The peeled waste was divided into two parts:
1) One part was converted into juice by blending and adding deionized
water before use in the MFC; and 2) One part was dried at 50 °C in an
oven for 24 h, ground into a powder, and homogenized. The physico-
chemical composition of the powder is given in Table 1. Both samples
were stored at 4 °C until further use. Anaerobic sludge for the MFC
anode was collected from the Sincheon wastewater treatment plant in
Daegu, South Korea. The sludge characteristics were as follows: chemi-
cal oxygen demand (COD) = 692.7 ± 7.5 mg/L; total organic compound
(TOC) = 242.8 ± 12.2 mg/L; pH = 6.45 ± 0.05; total suspended
solids = 16.4 ± 0.5 g/L; and volatile suspended solids = 9.5 ± 0.2 mg/L.
2.2. Assembly and operation of the MFC
A dual chamber laboratory scale MFC was used for this study, with
an anaerobic wastewater anode (OPW), and an aerobic air cathode (ox-
ygen) (Supplementary Fig. S1). A proton exchange membrane (PEM)
was used to separate the two chambers, thus avoiding intermixing of
the two solutions, and aiding transport of protons from the anode to
the cathode. Each electrode chamber measured 5 cm × 5 cm × 8 cm
(l × w × h), which equates to a volume of 200 mL. Platinum-coated
graphite cloth (20 wt.% Pt., 5 cm × 5 cm) was used as the cathode, and
graphite felt (5 cm × 5 cm, 3.18 mm thickness) as the anode. Nafion
117 (DuPont Co. USA) was used as a proton exchange membrane after
pre-treatment with boiling deionized water, aqueous H2O2 solution
(3% v/v), and dilute H2SO4 (0.5 M). This gave the highest power density
and coulombic efficiency (CE) of a dual chamber MFC reported to date
(Ghasemi et al., 2013). The anode and cathode were connected by insu-
lated copper wires with a fixed resistance of 500 Ω, unless stated
otherwise.
The MFC anode was inoculated initially with 20% v/v anaerobic con-
sortia and defined medium of the following composition (all measure-
ments are in mg/L): NaHCO3 = 420, MgSO4·7H2O = 200, (NH4)
2SO4 = 560, MnSO4·H2O = 20, and CaCl2 = 15, along with other
trace minerals and a buffer solution. Glucose was initially used as the
carbon source, and was later replaced by OPW. The medium in the
anode chamber was stirred continuously (magnetic stirrer) to maintain
a homogenous mixture, and the cathode chamber was aerated using an
air regulator. Stringent anaerobic conditions were maintained in the an-
odic chamber by introducing N2 (in anolyte) to every batch for 10 min,
and filling the anode chamber headspace with N2 gas using a pure nitro-
gen gas bag. Provisions were made in the MFC for sampling and for
inlet/outlet ports. The MFC was operated in batch mode in a
temperature-controlled compartment at 30 ± 2 °C. Samples were
Table 1
Characteristics of OPW powder.
Parameter Value (%)
Moisture 7.7 ± 0.20
Ash 4.4 ± 0.50
C 40.3 ± 0.02
H 5.8 ± 0.08
N 1.1 ± 0.20
S 0.1 ± 0.03
Protein 6.73 ± 1.26
Pectin 25.4
Cellulose 17.5
Hemicellulose 8.6
 W. Miran et al. / Science of the Total Environment 547 (2016) 197–205
withdrawn at regular intervals, filtered using a 0.45 μm filter, and sub-
jected to the desired analytical techniques.
2.3. Electrochemical calculations and chemical analysis
The potential difference, i.e., the cell voltage (V), between the anode
and cathode was measured every 3 min using a multimeter with digital
data acquisition system (Model 2700, Keithley Instruments Inc., USA)
connected to a personal computer to quantify the basic cell perfor-
mance. Current density (I) and power density (P) normalized to the
anode area (25 cm2) were calculated according to I = V/R and P =
V2/R, respectively, where R is the external resistance. Power and current
density curves were monitored to evaluate the maximum power using a
single cycle method. This method was based on the change of external
loads (Rext) during a single batch-fed cycle operating at its stable poten-
tial (Liu and Logan, 2004), where external resistance varied from 10 to
10,000 Ω. The internal resistance values (IRint) (equal to the external re-
sistance at maximum power density) were determined from the slope
of the current/voltage plots (Fan et al., 2007) according to Eq. (1):
V ¼ Ecell −IRint
where Ecell is the electromotive force. CE was evaluated by measuring
the ratio of total coulombs transferred to the anode from the substrate,
and the theoretical number of coulombs that can be transferred, and can
be calculated according to Eq. (2) (Logan et al., 2006):
Zt
8 Idt
0 Principal coordinate analysis (PCoA) and hierarchical clustering of bac-
CE ¼
FVan ΔCOD
where F is Faraday's constant (96,485 C), ΔCOD is the change in COD
over time (t) for each run, I is the average current, Van is the volume
of anolyte treated, and 8 is a constant used for COD. Suspended solids
(SS) and volatile suspended solids (VSS) were measured according to
standard methods (APHA, 2005). COD and TOC measurements were
carried out using a Hach spectrophotometer and TOC analyzer
(Shimadzu TOC-VCPH, Japan), respectively. Particle size distribution
(PSD) was measured using a Beckman Coulter LS 13320 particle size an-
alyzer. Protein content was calculated by multiplying the nitrogen con-
tent by the universal factor of 6.25. Pectin, cellulose, and hemicellulose
concentrations in orange peel were determined by thermogravimetric
analysis, using a Q600 TG/DTA analyzer, whereby the temperature
was increased from 20 to 900 °C at a rate of 10 °C/min under pure nitro-
gen. Pectinase and polygalacturonase (PG) activity were determined
using their respective substrates. Equal volumes of substrate (prepared
in citrate buffer, pH 4.8) and suitably diluted sample were incubated in a
water bath at 50 °C for 4 h. After incubation, a solution of dinitrosalicylic
acid (DNS, 2.5 mL) was added to quench the reaction, and the tubes
were allowed to stand in boiling water for 10 min. After cooling, the de-
veloped color was read at 575 nm using a UV–Vis spectrophotometer
(Agilent 8453, USA). The amount of reducing sugar released was quan-
tified using galacturonic acid as the standard. Enzyme activity was cal-
culated as the amount of enzyme required to release the measured
amount of galacturonic acid per mL of enzyme per minute under assay
conditions per gram of substrate used (Phutela et al., 2005).
2.4. Sampling, PCR amplification, pyrosequencing, and data analysis
Microbial community analysis was carried out for the initial inocu-
lated sludge fed into the MFC, the biofilm attached to the anode, and
the sludge suspended in the MFC. All sludge samples were centrifuged
at 10,000 rpm for 10 min and preserved at −80 °C. DNA was extracted
from the sludge samples for bar-coded pyrosequencing according to
manufacturer instructions, using a Fast-DNA kit (MPbio Solon, OH).
199
Primers Bac9F (5′-adaptor B-ACGAG TTT GAT CMT GGC TCA G-3′) and
Bac541R (5′-adaptor A-X-AC-WTT ACC GCG GCT GCT GG-3′) were
used for the amplification of bacterial 16S rRNA genes containing vari-
able regions (V1 to V3) — where “X” denotes unique 7- to 11-barcode
sequences inserted between the 454 Life Sciences adaptor A sequence
and the common linkers, AC and GA. All polymerase chain reaction
(PCR) amplifications were performed following preparation of the re-
quired solutions, according to specific cycling regimes (varied tempera-
tures and times) as described in the literature (Miran et al., 2015). The
resulting PCR products were purified for subsequent pyrosequencing
using a PCR purification kit (Solgent, Korea). Quantification of the puri-
fied PCR products was achieved using an enzyme-linked immunosor-
bent assay reader, equipped with a Take3 Multi-Volume Plate.
Pyrosequencing was performed by Macrogen (Seoul, Korea) using a
454 GS-FLX Titanium platform (Roche, Basel, Switzerland) and the
resulting sequencing reads were processed to remove sequencing
noise as described previously (Lee et al., 2012). The denoised sequenc-
ing data were analyzed using the Ribosomal Database Project (RDP) py-
rosequencing pipeline (http://pyro.cme.msu.edu/). Pyrosequencing
reads were assigned to specific samples based on their unique barcodes,
ð1Þ and the barcodes subsequently removed. Only pyrosequencing reads
with fewer than two “N″ (undetermined nucleotides) and N300-bp
(bp = base pair) read lengths were selected for subsequent analysis.
Operational taxonomic units (OTU) and rarefaction curves were gener-
ated using the RDP pyrosequencing pipeline at a 3% dissimilarity level.
The Shannon-Weaver and Chao1 biodiversity indices and evennesses
were also calculated using the RDP pyrosequencing pipeline. Taxonomic
assignment of the high quality bacterial reads was performed using the
RDP naive Bayesian rRNA Classifier using 80% confidence thresholds.
ð2Þ
terial communities were carried out using UniFrac analysis (Lozupone
and Knight, 2005).
3. Results and discussion
3.1. Startup and voltage generation
The dual chamber MFC was initially acclimatized with anaerobic
sludge and glucose as the sole carbon source. Once this preliminary ac-
climatization (i.e., exoelectrogens growth on the anode) was complete
(defined by the production of a stable voltage), a series of batch exper-
iments were carried out using the OPW. Powdered OPW, unfiltered
juice, and the filtered medium (supernatant) were fed into the MFC to
Fig. 1. Voltage generation from OPW (powdered, unfiltered, and filtered) at an external
resistance of 500 Ω.
200 W. Miran et al. / Science of the Total Environment 547 (2016) 197–205
monitor its performance in terms of voltage production, as illustrated in
Fig. 1. Bioelectricity generation began rapidly, with the maximal stable
voltage achieved within 2 h in all cases (batches I, II, and III). The dura-
tion of sustained voltage varied with the initial concentrations in all
batches (the feed was diluted to b1500 mg/L TCOD for all batches).
The effect of powdered peel waste concentration (in a defined medium)
was investigated at 0.5, 1, and 1.5 g/L. In this case, the average voltage of
the MFC during the stable voltage generation phase was 0.53 ± 0.02 V
with an external resistance of 500 Ω. The maximum voltage for the un-
filtered medium-fed batches was slightly higher, i.e., 0.55 ± 0.02 V. It
was previously reported that use of a medium-containing solid sub-
strate gave limited power production due to slow hydrolysis of the par-
ticulate material. This demonstrated that particle size is also important
with regards to maximum voltage generation (Rezaei et al., 2009a). In
the current method, degradation and size reduction of the orange peel
powder took place (Supplementary Fig. S2), which may aid in high cu-
mulative power production. Compared to the unfiltered medium, the
voltage obtained for the filtered medium was approximately 7% higher
(i.e., 0.59 ± 0.02 V). A higher soluble COD (SCOD) in the substrate may
be beneficial for the MFC operation, providing easily accessed carbon
sources, nutrients, and supplements to the electrogens (Min et al.,
2005). The electrochemical behavior of the fed-batch MFC was reason-
ably stable over all cycles, as there was little change in the maximum
voltage production at the various initial concentrations, with only a var-
iation in cumulative power taking place. These preliminary results indi-
cate that anodic microorganisms can utilize carbon matter present in
OPW to release electrons, i.e., they generate bioelectricity from the
OPW. However, the key task is to get MFC technology out of the labora-
tory and develop efficient systems for power generation at industrial
levels. There are recent progresses in novel types of electrodes, under-
standing of the influence of membranes and separators is far better,
and outcomes from numerous pilot-scale studies are all good indicators
that commercialization of the MFC technology could be possible very
soon now (Hernandez-Fernandez et al., 2015; Logan, 2010).
3.2. Polarization/power density curves and internal resistance
Polarization and power density curves for different forms of OPW
were obtained using stable batch-fed MFCs, by decreasing the external
Fig. 2. Power output and power density curves of MFC using different forms of OPW.
resistance from 10,000 to 10 Ω (Fig. 2). The maximum power density
achieved for the dry powder fed-batch was 277.5 ± 5.3 mW/m2, corre-
sponding to a current density of 745.0 ± 7.07 mA/m2. In comparison,
maximum power densities for the unfiltered peel waste juice and fil-
tered medium were 320.5 ± 22.6 mW/m2 (current density =
800.0 ± 28.3 mA/m2) and 358.8 ± 15.6 mW/m2 (current density =
847.0 ± 18.4 mA/m2), respectively. These results are a significant im-
provement on other agriculture based biomasses used in MFCs, includ-
ing rice straw and wheat straw substrates (Hassan et al., 2014; Zhang
et al., 2009). However, it is not always realistic to compare the maxi-
mum power densities among various MFC systems, due to variation in
MFC conditions. Such variables include electrode metallurgy, the type
of proton exchange membrane, the distances between electrodes, and
the type and concentration of the electrolyte and the electron donor
(Kim and Lee, 2010). For example, the redox potential of the substrates
alters the current generation capability based on the difference between
the substrate and anode, giving information on whether or not the
redox process is thermodynamically favorable for the recovery and
transfer of electrons. In all cases, the internal resistance of the OPW-
fed MFC was approximately 237.3 ± 21.0 Ω. This resistance value indi-
cates the ease by which electrons flow from the anode to cathode, and
ultimately, the current generation from the system. In addition to a suit-
able MFC substrate and electrolyte/electrode spacing, the anode biofilm
can also play a significant role in decreasing the internal resistance of
the cell (Ramasamy et al., 2008), and can increase power production.
3.3. Removal of organics and columbic efficiency
The performance characteristics of the dual chamber MFC operated
using different forms of OPW were studied in terms of COD and TOC re-
moval (Fig. 3). The maximum COD removal for dry powder peel waste
was 78.3% at 1217.0 ± 33.9 mg/L (corresponding to 698 ± 52.3 mg/L
soluble COD). This value was 14.6% higher compared to batches with
an initial COD of 440.0 ± 13.4 mg/L (255.0 ± 8.5 mg/L soluble COD).
COD and TOC removal was N75% for batches II and III, with the highest
removal obtained in filtered peel waste-fed batches (N 80% COD and
TOC removal). These results indicate the importance of the MFC system
not only for bioelectricity generation from OPW, but also for their ability
to reduce the environmental burden. Based on current generation and
 W. Miran et al. / Science of the Total Environment 547 (2016) 197–205
Fig. 3. COD and TOC removal in batches fed with a) powdered, b) unfiltered, and c) filtered
peel waste.
COD removal, a CE of 15.50% was obtained for filtered batch OPW, which
was higher than for powdered (7.55%) and unfiltered OPW (11.92%)
batches. CE was limited by several factors, including electron consump-
tion by methanogenesis, aerobic respiration of the cathode biofilm, and
oxygen crossover (Liu et al., 2005). Moreover, it also indicates that even
with high COD removal, a limited portion of the organic matter could be
captured as current.
201
3.4. Microbial community structures
The microbial community structures of the initial inoculum (S1),
anode-attached biofilm (S2), and bacteria in the anodic chamber sus-
pension (S3) were analyzed based on 16S rRNA gene pyrosequencing.
Pyrosequencing is advantageous, as it can identify both the dominant
OTUs and the rarer communities. The total numbers of sequences ob-
tained were 1602, 2119, and 1309, which reduced to high quality
reads of 1416, 1845, and 1155 after trimming, for S1, S2, and S3, respec-
tively (Table 2). The OTUs obtained by each community (S1, S2, and S3)
were 407, 461, and 258, respectively. A slight increase in the Shannon
diversity index (H′) was observed in S2 (4.77) when compared to S1
(4.72), while a decrease in S3 was observed (4.03). The difference in
bacterial community structures was identified based on their phyloge-
netic lineages by weighted Fast UniFrac analysis. Principal coordinates
analysis showed a significant difference between the S1, S2, and S3 com-
munities (Fig. 4a). However, the S2 and S3 communities were close in
comparison to S1, as demonstrated by dendrogram cluster analysis
(Fig. 4b). Indeed, it was previously reported that communities devel-
oped in MFCs can change significantly from the initial inoculum, mainly
due to different environments, and the degree of substrate complexity
(El-Chakhtoura et al., 2014).
The communities belong to 13, 15, and 11 phyla in the cases of S1, S2,
and S3, respectively. The major phyla in S1 were Proteobacteria (64.5%),
Bacteriodetes (13.0%), and Chloroflexi (11.0%) (Fig. 5a). However, in both
S2 and S3, the most dominant phylum was firmicutes (31.0% and 43.2%,
respectively). The number of unclassified bacteria at phyla level were
highest in S2 (22.2%), followed by S3 (14.8%) and S1 (6.21%), herby indi-
cating the highest diversity was in the anode-attached biofilm. At the
genera level, the most dominant identified genera in the anode biofilm
were Enterococcus (18.1%), Paludibacter (6.1%), and Pseudomonas (3.1%)
when compared to the initial inoculum, where the dominant genera
were Brevundimonas (26.1%), Stenotrophomonas (9.2%), and
Psychrobacter (7.8%). The dominant genera in the anode chamber sus-
pension were Enterococcus (19.3%), Thiomonas (4.17%), and Petrimonas
(3.14%). Full details of the genera identified (content N 0.1%) in all
three samples are shown in Fig. 5b. The quantities of unclassified genera
in S1, S2, and S3 were 31.4, 56.6, and 63.7%, respectively. The high num-
bers of unclassified genera in S2 and S3 demonstrate the remarkable di-
versity of the microbial communities in both the anode biofilm and in
the anode suspension. Unsaturation of the rarefaction curves (Supple-
mentary Fig. S3) also indicates the high diversity of the microbial com-
munity, which depends not only on the initial inoculum, but also on the
organic source used by the microorganisms. This organic source may be
directly used by exoelectrogens, or may be used by fermentative bacte-
ria following the production of metabolites, and used later by
exoelectrogens (El-Chakhtoura et al., 2014). With reference to
current-producing bacteria, Pseudomonas is a well-known
exoelectrogen found in the studied anode biofilm. Other well-known
exoelectrogens, such as Geobacter and Shewanella were not detected.
As OPW is likely to undergo fermentative degradation, fermentative
bacteria possibly played an important role in the overall MFC perfor-
mance. Enterococcus was the most dominant genus in the anode biofilm
and in the anode chamber suspension, and its enriched form was also
examined recently as a self-mediated exoelectrogens for successful cur-
rent generation in MFCs (Wu et al., 2013). This genus may have played a
role in current generation in our study, but it is also believed to have a
fermentative nature. Other predominant fermentative bacteria detected
in the anode suspension and biofilm were Petrimonas, Acinetobacter, and
Acetobacterium. It can also be assumed that a large number of unclassi-
fied genera may have played a significant role in OPW degradation and
current generation. Overall, the combination of exoelectrogens, fermen-
tative bacteria, and other functional bacteria is important for the effec-
tive degradation of substrates and for bioelectricity production.
Studies are underway in this very important area for enhancing MFC
performance, including identification of bacterial communities and
202 W. Miran et al. / Science of the Total Environment 547 (2016) 197–205

Table 2
Summary of pyrosequencing data and statistical analyses of bacterial communities in initial inoculum, biofilm attached with anode electrode, and bioflora in anode compartment
suspension.

Sample type Initial inoculum Biofilm attached with anode electrode Bioflora in anode compartment suspension

Total sequences 1602 2119 1309

Sequences after trimming 1416 1845 1155
Average sequence length after trimming 456 453 452
Standard deviation of sequence length 43.81 51.34 57.36
OTU 407 461 258
Shannon-weaver index (H′) 4.72 4.78 4.03
Chao 1 index 866.04 999.39 534.85
Evenness 0.787 0.779 0.726
No. of phyla 13 15 11
No. of genera 43 51 35

their optimal combination for achieving simultaneous higher power
outputs and degradation of utilized substrates.
3.5. Enzyme analysis and current generation by pectin and cellulose
As cellulose and pectin are the major (polysaccharide) components
of orange peel powder, their degradation may play a significant role in
current generation. Therefore, pectin and cellulose were tested individ-
ually in the MFC for current generation at a concentration of 1 g/L. Fig. 6a
shows the tendency of MFC microbes to generate bioelectricity from
pectin in significantly larger quantities than from cellulose. Cellulose is
a linear homopolymer of (1–4)-linked β- -glucopyranose, which forms
a macromolecule with a highly stable crystalline lattice structure. As
such, cellulose is highly resistant to attack by microbial/enzymatic

Fig. 4. a) PCoA results, and b) hierarchical clustering of bacterial communities, showing the relationships between initial inoculum, biofilm attached with anode electrode, and bioflora in
anode compartment suspension.
 W. Miran et al. / Science of the Total Environment 547 (2016) 197–205 203

Fig. 5. Taxonomic classification of pyrosequences at a) phylum and b) genus levels of initial inoculum, biofilm attached with anode electrode, and bioflora in anode compartment
suspension.

agents and its hydrolysis is slow (Hong and Sun, 2013). Glucose is con-
sidered the most potent inhibitor in the hydrolysis of cellulose to glu-
cose. In MFCs, glucose can also be degraded, resulting in electricity
generation, which can improve the overall hydrolysis of cellulose to glu-
cose. When cellulose is the substrate, enzymes such as cellulase can be
added externally to enhance the hydrolysis rate and improve the overall
MFC performance in terms of cellulose and COD removal, along with an
increase in power density and coulombic efficiency (Rezaei et al., 2008).
Similarly, external enzymes can be used to enhance OPW performance
or other agriculture-based MFCs. It is important to identify that inocu-
lum source used in this study was taken from wastewater treatment
plant (considered as a good source of exoelectrogens and mixed cul-
tures for improved MFC performance), as it has also been used by
other researchers for agriculture byproduct substrates in MFCs (Zhang
et al., 2013). However, wastewater treatment inoculums may lack cellu-
lose degrading capability as in this study. Therefore, bioflora from native
source like orange waste vicinity may be helpful in degrading cellulose
as well but ideal combination would be mixtures of exoelectrogens and
cellulose degrading bacteria for enhancing overall performance of cellu-
lose containing substrates in MFCs. As significant current generation
was observed from pure pectin degradation, enzymes commonly asso-
ciated with pectin degradation, (i.e., pectinase and polygalacturonase)
were investigated with the OPW dry powder batch (1 g/L). The maxi-
mum enzyme concentrations obtained were 18.55 U/g and 9.04 U/g of
pectinase and polygalacturonase, respectively (based on substrate
mass) (Fig. 6b). The predominant pectin-degrading bacteria identified
204 W. Miran et al. / Science of the Total Environment 547 (2016) 197–205
Fig. 6. a) Voltage generation by pure pectin and cellulose. b) Pectinase and
polygalacturonase activity during voltage generation in the MFC fed with OPW.
in both the suspension and the anode biofilm, which may be the source
of these enzymes, belong mainly to the Clostridium and Bacteroides
genera.
4. Conclusions
Bioelectricity was successfully generated using orange peel waste
(OPW) as a renewable carbon source in a dual chamber mediator-less
microbial fuel cell (MFC). High reproducibility in terms of voltage gener-
ation (max. = 0.59 ± 0.02 V at 500 Ω) was achieved for batches fed
with different forms of OPW (namely powdered, filtered, and unfiltered
peel waste). Microbial community analysis by high-throughput pyrose-
quencing revealed that a significant difference existed between the bac-
terial communities present in anode biofilm and in the anode
suspension compared to the initial inoculum. The combination of fer-
mentative microorganisms and exoelectrogens aided in achieving effec-
tive OPW degradation combined with high current generation.
Pectinase and polygalacturonase activity was detected in MFCs due to
the degradation of pectin present in OPW. Thus the MFC can be practi-
cally used as an alternative energy technology using the agricultural
byproduct (OPW) as an organic substrate, but it needs to be further im-
proved in order to make it competitive with alternative energy technol-
ogies. For instance, capital cost need to be decreased by applying single
chamber membrane less MFCs (or other modified designs) and power
generation need to be increased by right combination of microbes.
Acknowledgments
This work was supported by the Human Resource Training Program
for Regional Innovation and Creativity through the Ministry of Educa-
tion (ME) and National Research Foundation (NRF) of Korea (NRF-
2014H1C1A1066929). This study was also supported by grants NRF-
2013R1A1A4A01008000 and NRF-2009-0093819 through the ME and
NRF of Korea. This research was also supported by the NRF grant by
the Korea government (MSIP) (NRF-2015M2A7A1000194).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2016.01.004.
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