Bioresource Technology 100 (2009) 5132–5139

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

Electricity generation using chocolate industry wastewater and its treatment

in activated sludge based microbial fuel cell and analysis of developed
microbial community in the anode chamber
Sunil A. Patil a, Venkata Prasad Surakasi b, Sandeep Koul a, Shrikant Ijmulwar a, Amar Vivek a,
Y.S. Shouche b, B.P. Kapadnis a,*
a
Department of Microbiology, University of Pune, Pune 411 007, Maharashtra, India
b
Molecular Biology Unit, National Centre for Cell Science, Pune 411 007, Maharashtra, India

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

Article history: Feasibility of using chocolate industry wastewater as a substrate for electricity generation using activated
Received 14 March 2009 sludge as a source of microorganisms was investigated in two-chambered microbial fuel cell. The max-
Received in revised form 18 May 2009 imum current generated with membrane and salt bridge MFCs was 3.02 and 2.3 A/m2, respectively, at
Accepted 19 May 2009
100 X external resistance, whereas the maximum current generated in glucose powered MFC was
Available online 17 June 2009
3.1 A/m2. The use of chocolate industry wastewater in cathode chamber was promising with 4.1 mA cur-
rent output. Significant reduction in COD, BOD, total solids and total dissolved solids of wastewater by
Keywords:
75%, 65%, 68%, 50%, respectively, indicated effective wastewater treatment in batch experiments. The
Microbial fuel cell
Chocolate industry wastewater
16S rDNA analysis of anode biofilm and suspended cells revealed predominance of b-Proteobacteria
Activated sludge clones with 50.6% followed by unclassified bacteria (9.9%), a-Proteobacteria (9.1%), other Proteobacteria
Wastewater treatment (9%), Planctomycetes (5.8%), Firmicutes (4.9%), Nitrospora (3.3%), Spirochaetes (3.3%), Bacteroides (2.4%)
16S rDNA microbial analysis and c-Proteobacteria (0.8%). Diverse bacterial groups represented as members of the anode chamber
community.
Ó 2009 Published by Elsevier Ltd.

1. Introduction The pure microbial cultures have limitations for technical appli-
cation because of necessity for highly sterile conditions, culture
Use of fossil fuels has contributed to global climate change, degradation and high cost. Mixed cultures or microbial consortia
environmental degradation and health problems. In this context, have been shown to be robust and more productive than single
many researchers predict that biohydrogen and bioelectricity strains and their extraction can be easily achieved from natural
could play an important role as fuel in the near future. Biological sources (Ha et al., 2008). The microbial communities in MFCs with
hydrogen and electricity could team to provide attractive options different types of substrates and microbial sources have been ana-
in transportation and power generation. Microbial fuel cell (MFC) lyzed to study the involvement of microorganisms in electricity
technology offers the possibility of harvesting electricity from or- generation (Holmes et al., 2004; Jung and Regan, 2007). Several
ganic waste and renewable biomass. It has been developed as a no- groups have characterized active microbial communities at anode
vel technique to gain energy with simultaneous wastewater to potentially improve MFC performance by addressing microbial
treatment. Efforts have been dedicated by researchers towards constraints. Various phylogenetically diverse bacteria are reported
the development of MFCs for the electricity generation in last dec- to generate electricity in MFCs (Logan and Regan, 2006; Ha et al.,
ade. Various carbohydrates and industrial wastewaters have been 2008). However, there is much scope for improvement in the per-
tried for production of electricity (Catal et al., 2008; Liu et al., formance of MFCs using microorganisms from natural habitats.
2004). The MFC research is strongly growing with respect to alter- Due to environmental concerns, there is a great interest in
native substrates, potent microbial cultures, electrodes, ion ex- developing new methods either to reduce the treatment costs or
change membranes and anodic electron transfer mechanisms to get value added products from wastes. The MFC technology
(Schroeder, 2007). proved to be a novel approach for treating wastewater with simul-
taneous production of electricity (Venkata Mohan et al., 2008). The
chocolate manufacturing industry wastewater can be character-
* Corresponding author. Tel.: +91 20 25690643; fax: +91 20 25690087.
ized as nontoxic because it is devoid of hazardous compounds,
E-mail address: [email protected] (B.P. Kapadnis). but with high content of total solids (TS), biochemical oxygen

0960-8524/$ - see front matter Ó 2009 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2009.05.041
 S.A. Patil et al. / Bioresource Technology 100 (2009) 5132–5139 5133

demand (BOD) and chemical oxygen demand (COD). To our 2.4. Testing of different catholytes in the membrane MFC
knowledge, there are no reports on electricity generation using
chocolate industry wastewater with microbial consortia from Different catholytes viz. K3[Fe(CN)6], chocolate industry waste-
natural habitats. Therefore, in the present work the chocolate water and phosphate buffered air cathode were tested in the cath-
industry wastewater was investigated as a substrate for generation ode chamber and performance of the MFCs was studied with
of electricity in both salt bridge and proton exchange membrane respect to these catholytes. Chocolate industry wastewater and
MFCs using activated sludge as a source of microorganisms. The activated sludge were used in the anode compartments of all these
electricity generated using wastewater was compared with that MFCs. The electrodes used in cathode chamber were graphite rods.
of glucose as a substrate. The use of same wastewater was tried The ferricyanide (100 mM) was prepared in 100 mM KH2PO4 buffer
as a catholyte in the cathodic chamber and performance was com- (pH 7.5). Phosphate buffer (10 mM KH2PO4) was used in air cath-
pared with air cathode and K3[Fe(CN)6]. Phylogenetic analysis of ode experiments and it was continuously sparged with air applying
microbial communities from anode chamber of the MFC was car- sparger. In case of chocolate industry wastewater as catholyte, the
ried out to study the microbial diversity. cathode chamber was also sparged with air. These experiments
were carried out with membrane MFC and other experimental con-
ditions were same as mentioned earlier.
2. Methods
2.5. Analysis of various parameters of chocolate industry wastewater
2.1. Substrate and source of microorganisms
The characteristics of chocolate industry wastewater before
The chocolate industry wastewater was collected from the using it in MFC are presented in Table 1. Various parameters of
Chocolate India Malted Food Division, Warananagar (India). The wastewater viz., pH, COD, BOD, total solids, total dissolved solids,
source of microorganisms was activated sludge from Municipal acidity and electrical conductivity, were experimentally deter-
wastewater treatment plant, Kolhapur, India. All chemicals (ana- mined before and after experimental runs using standard methods
lytical or biochemical grade) and other materials used in MFC (APHA, 1995) to monitor progress of wastewater treatment. This
experiments were purchased from Merck and SD fine. study was conducted with the samples from proton exchange
membrane MFC. All experiments were repeated and the mean val-
2.2. Experiments using salt bridge and membrane MFCs ues were considered for the all calculations.

The two-chambered MFCs were used throughout the study. The 2.6. Phylogenetic analysis of microbial communities from anode of
experiments were carried out in batch mode using salt bridge and MFC
proton exchange membrane MFCs composed of two 400 ml volume
bottom flasks. The salt bridge MFC was constructed by joining two 2.6.1. DNA extraction
bottles with glass tube (5 cm length; 0.6 cm inner diameter) pre- The DNA was extracted from the scraped anode biofilm and sus-
pared using agar and KCl powder (1:1). In proton exchange mem- pended cells from anode chamber (at the completion of a batch cy-
brane MFC, membrane (Nafion-117) of 2.5 cm diameter was cle) of MFC using protocol explained earlier (Yeates et al., 1997).
clamped between anode and cathode compartments. The electrodes Some minor modifications were introduced such as cell pellet of
used were graphite rods (projected area of 16.485 cm2). The anode 2 ml enrichment culture was suspended in extraction buffer
part of the both MFCs was filled with 200 ml chocolate industry (100 mM Tris–HCl (pH 8.0), 100 mM Na2EDTA (pH 8.0) with
wastewater as substrate and 20 ml activated sludge as microbial 20 mg/ml lysozyme (Sigma–Aldrich Co., USA) and incubated for
inoculum, obtained from previously enriched MFC to reduce the 2 h at 37 °C with continuous shaking. Proteinase K (Invitrogen,
start up time. The 100 mM K3[Fe(CN)6] prepared in 100 mM KH2PO4 USA) was added to a final concentration of 100 lg/ml and incu-
buffer (pH 7.5) was used in the cathode chamber of both MFCs. The bated further at 55 °C for 2 h with continuous shaking. The NaCl
anode chamber and cathode chamber with ferricyanide as catholyte (0.5 M) was added and incubated at 72 °C for 30 min. Subsequently
were maintained at anoxic conditions. The voltage generated in DNA was extracted by phenol and chloroform:isoamyl alcohol
MFCs was measured by using a voltammeter with an external resis- (24:1) method as per Sambrook et al. (1989). DNA pellet was
tor (100 X) connected across the electrodes and recorded after reg- washed with 70% ethanol. The product was dissolved in TE buffer
ular time intervals. The voltage values were recorded only after a (pH 8.0).
steady state. Current (i) was calculated using relationship, i = V/R,
where V is the voltage and R is resistance. Power (P) was calculated 2.6.2. Amplification, cloning, and sequencing of 16S rDNA
using the relationship, P = iV, and normalized by the surface area of The 16S rDNA was amplified from extracted DNA using univer-
the anode. All experiments were conducted at 28 ± 2 °C. sal bacterial primers 530F and 1490R as described previously
(Wani et al., 2006). In the above PCR reactions, the number of
2.3. Electricity generation using synthetic medium with glucose as cycles was reduced to 25 so as to minimize PCR bias. The PCR
substrate

These experiments were conducted with proton exchange Table 1

membrane MFC using synthetic medium containing glucose as a Chocolate industry wastewater parameters before and after feeding the MFC.

substrate instead of chocolate industry wastewater, with activated Parameter Parameter

sludge in the anode chamber. The ingredients of medium included Before After
(g/l): KCl (0.74), NaCl (0.58), KH2PO4 (0.68), K2HPO4 (0.87), NH4Cl
pH 5.76 6.35
(0.28), MgSO4
7H2O (0.1), CaCl2
2H2O (0.1), and to this, 1 ml of COD (mg/l) 1459 368
trace element mixture was added (Zehnder et al., 1980). We used BOD3 at 27 °C (mg/l) 640 230
2.0 g/l glucose as a carbon source in the synthetic nutrient med- Total solids (mg/l) 2344 754
ium. The pH of the medium was adjusted to 6.8. The other exper- Total dissolved solids (mg/l) 404 170
Acidity (mg/l) 0.388 0.582
imental conditions were same as mentioned earlier for previous
Electrical conductivity (lmhos/cm) 0.44 0.51
experiments.
5134 S.A. Patil et al. / Bioresource Technology 100 (2009) 5132–5139

products were purified using a PCR purification kit (Qiagen, USA).

Clone libraries of PCR products were constructed using the TOPO
TA Cloning Kit (Invitrogen, USA) inoculated with plasmid pCR2.1.
Successful transformants were inoculated into Luria–Bertani broth
containing 100 mg/ml ampicillin and were allowed to grow over-
night. Clones containing putative 16S rRNA genes were screened
by PCR amplification using M13 primers and were visualized on
a 1% agarose gel to ensure the presence of inserts of the expected
size. Clones that contained no insert or inserts of incorrect size
were excluded from sequencing. Sequencing was performed on
3730 DNA analyzer (Applied BioSystems, USA) using the ABI Big-
Dye version 3.1 sequencing kit as per the manufacturer’s instruc-
tions. The sequences of cloned inserts were deposited in GenBank
under accession numbers FJ624158 to FJ624199.

2.6.3. Phylogenetic analysis

Sequences were compared to a current database of rRNA gene
sequences from GenBank (July, 2008), and reference sequences
were chosen based on BLAST similarities. Sequences were also Fig. 1. Voltage (V) at 100 X in: (A) proton exchange membrane MFC, (B) salt bridge
compared to the current database at Ribosomal Database Project MFC (both with chocolate industry wastewater and activated sludge in the anode
(RDP) using the RDP query program (Maidak et al., 1999). All se- chamber) and (C) proton exchange membrane MFC with synthetic medium
(glucose) and activated sludge in the anode chamber. The catholyte was common
quences were checked for possible chimeric artifacts by Mallard
in all these experiments.
program (Ashelford et al., 2006). Predicted chimers were further
analyzed by using Pintail (Ashelford et al., 2006) and Bellerophon
(Huber et al., 2004). Multiple sequence alignments were performed and 15 days until a stable current generation was obtained (Liu
using CLUSTAL W, Version 1.8 (Thompson et al., 1994) and RDP et al., 2005). This might be the reason for the generation of signif-
alignment program (Maidak et al., 1999). Multiple sequence align- icant voltage after 5–6 days. However, we speculate that the bio-
ment was edited and corrected manually using DAMBE (Xia and film formation took less time as higher voltage was observed
Xie, 2001) to get unambiguous sequence alignment. Appropriate after 5 days in case of all experiments. This is because of the use
subsets of 16S rDNA sequences were selected on the basis of initial of previously enriched microbial sample. The rapid increase of volt-
results and subjected to further phylogenetic analysis using the age might be because of the presence of components in chocolate
neighbor-joining method implemented through DNADIST from industry wastewater that are easily utilized by anodic bacteria.
PHYLIP version 3.61 (Felsenstein, 1993). The OTUs (Operational After subsequent use of substrate, the voltage began to decrease
Taxonomic Units) were generated by using the DOTUR program in batch experiments. The maximum current density obtained
(Schloss and Handelsman, 2005) with furthest neighbor algorithm. was 3.02 A/m2 [4.98 mA; 1.5 W/m2] and 2.3 A/m2 [3.9 mA;
OTUs with identity P97% were used for taxonomic assessment. 0.94 W/m2] with membrane microbial fuel cell and with salt bridge
Rarefaction was used to evaluate OTU richness of the clone li- MFC, respectively, in batch experiments. The results obtained with
braries. Shannon diversity was used to evaluate OTU evenness. salt bridge MFC were lower in comparison to membrane MFC,
The Chao1 richness estimator was used to estimate the total num- which can be attributed to more internal resistance of the system
ber of species in our samples. A neighbor-joining phylogeny was (Logan et al., 2006). But the use of proton exchange membrane is
inferred by using MEGA3 (Tamura et al., 2007). It was constructed expensive and economically unsustainable, though the outputs
with a distance matrix calculated according to Kimura’s two- are greater with membrane MFC.
parameter model (Kimura, 1980) and Jukes Cantor model (Jukes
and Cantor, 1969). Bootstrap values were obtained from 1000 ran- 3.2. Electricity generation with glucose in synthetic medium
dom sampling.
Glucose, as it is readily utilizable substrate was used in syn-
thetic media for the comparison studies with the chocolate indus-
3. Results and discussion try wastewater in membrane MFC. The maximum voltage obtained
with synthetic media was 0.514 V after 159 h which was higher as
3.1. Electricity generation in salt bridge MFC and proton exchange compared to chocolate wastewater as substrate (0.498 V after
membrane MFC 177 h). The voltage generated with synthetic media containing glu-
cose is greater comparable to wastewater under similar set of
The current generation is based on microbial electron transfer experimental conditions (Fig. 1). The maximum current density
at the graphite electrodes in case of both salt bridge and membrane obtained with glucose was 3.1 A/m2 [5.14 mA; 1.6 W/m2] with
MFCs. Fig. 1 shows the comparison of the voltage generated in both membrane MFC. The current generation from all these MFCs con-
MFCs. The maximum voltage generated was 0.398 V after 135 h in siderably decreased during 6–12 days. The decrease in voltage
case of salt bridge MFC, whereas it was 0.498 V after 177 h in pro- was rapid in case of glucose powered MFC than chocolate industry
ton exchange membrane MFC (Fig. 1). Initially after microbial sam- wastewater based MFCs. A clear pattern of decreasing current pro-
ple inoculation, a sudden rise in the voltage was observed. This duction levels was observed, though the rate of decrease was
immediate voltage fluctuation that was obtained might be due to slower after 12th day in case of chocolate wastewater based fuel
both chemical and biological factors based on the difference of cells. This decrease is most likely due to a depletion of substrate
the voltage between the two chambers. The voltage values were in the anode. The results indicate that it is more feasible to use
recorded only after reaching steady state. The time taken to gener- industrial wastewaters as it showed good voltage and which was
ate the maximum voltage by salt bridge MFC was less as compared constant for more period in comparison to synthetic medium, the
to the membrane MFC at the similar set of experimental condi- use of which is uneconomical. We did not generate a polarization
tions. The biofilm formation has been reported to take between 6 curve for our experiments and this means that the maximum
 S.A. Patil et al. / Bioresource Technology 100 (2009) 5132–5139 5135

voltage and current output reported may not be the maximum lyzed (Table 1). The remarkable changes in the values of various
achievable with our system. parameters of wastewater were observed. The pH of the wastewa-
ter was increased to 6.3 from pH 5.76 after the batch experiments.
3.3. Testing of different catholytes The 75% and 64% reduction in the values of COD and BOD of waste-
water was observed, respectively, with the membrane MFC. The
The maximum voltage (0.498 V) was generated with ferricya- decrease in total solid content (68%) and total dissolved solids
nide as electrolyte in cathode chamber. The use of chocolate indus- (58%) signifies the use of it by the microorganisms and indicates
try wastewater as catholyte showed greater results (0.412 V) in further wastewater treatment. The increase in the conductivity
comparison to air cathode with 10 mM K2HPO4 (0.311 V) in batch (16%) of anodic sample was observed after the experimental run
experimental runs. The maximum current output obtained was which might be due to the presence of electrochemically active
3.1 mA [1.88 A/m2; 0.58 W/m2] and 4.1 mA [2.49 A/m2; 1.02 W/m2] microorganisms. It is proposed that with continuous operation of
with air cathode and chocolate industry wastewater as catholyte, MFC it could be possible to increase the wastewater treatment effi-
respectively (Fig. 2). We observed ferricyanide [4.9 mA; 3.02 A/ ciency with simultaneous electricity generation.
m2; 1.5 W/m2] as good candidate in cathode, which has been al- The MFC technology has limitations in implementing it for
ready proved as an efficient catholyte. But it has limitations in wastewater treatment, as the current outputs are still relatively
use because of expenses and need of its continuous replacement lower and the technology is not yet in commercialized phase.
in MFCs. The use of air cathode has been tried by various research- Based on the voltage difference between the electron donor and
ers showing significant results (Liu and Logan, 2004; Venkata Mo- acceptor, a maximum voltage of 1 V can be expected in MFCs
han et al., 2008). Compared to air cathode MFC, we observed (Liu et al., 2004), which is greater than the 0.5 V that was produced
more current output in case of chocolate wastewater catholyte in our study. Currents and power outputs are lower than what is
based MFC. This is indicative of involvement of some additional fac- theoretically possible. However, by linking several MFCs together,
tors (chemical or microbial) from wastewater in increased current the voltage can be increased. To make MFCs feasible for wastewa-
output, which needs to be worked out and addressed. These results ter treatment, the construction and operating costs must be re-
indicated the feasibility of using wastewaters as catholytes, though duced. In addition, the rate of electron transport must be
additional studies are to be carried out for the effective exploitation improved which may be achieved by selecting a well-adapted
of wastewaters in cathode chambers. The use of catalyst or an elec- anodophilic microbial community and optimizing the MFC operat-
tron mediator in abiotic cathode increases the cost and lowers the ing conditions. Higher voltages might be expected in this system
operational sustainability. Such disadvantages can be overcome with more concentrated wastewater. The use of same wastewater
by biocathodes which make use of microorganisms to assist catho- in cathode chamber also yielded good output. The use of wastewa-
dic reactions (He and Angenent, 2006). The wastewater showed ters can be considered effective as catholytes in MFCs as they re-
comparatively good electricity output, which can be considered as duce costs in comparison to ferricyanide based MFCs. The
biotic cathode in which microbial activities might be involved in chemical and/or microbial community analysis from wastewater
cathodic electrode reduction. Also using bacteria in the cathode can be helpful to understand the microbial reactions occurring at
compartment provides the opportunity to render the process more cathode and their role in increased current output.
sustainable and cost efficient, as no catalyst addition to the system
is needed. The industrial wastewater use in cathode might be sig- 3.5. Analysis of microbial communities in anode chamber
nificant as it does not add to the cost and yielded significant output.
Further studies on microbial communities and activity are neces- Nearly 300 clones were recovered and resulted in 241 high
sary to understand the cathodic electrode reduction. quality sequences. Approximately 950 bp of bacterial 16S rDNA
fragments (530–1490 region of Escherichia coli) were obtained.
3.4. Wastewater treatment The fragments contain variable and conserved regions that accu-
rately reflect the phylogenetic position of the corresponding clone
Various parameters of chocolate industry wastewater from the library (Lane et al., 1985). The distribution of bacterial clones with
anodic chamber before and after the experimental run were ana-

Fig. 2. Effect of different catholytes on electricity generation studied with proton

exchange membrane MFC. In all experiments the anode chamber was with Fig. 3. Distribution of bacterial clones in proton exchange MFC enriched with
chocolate wastewater and activated sludge. activated sludge and chocolate industry wastewater.
5136 S.A. Patil et al. / Bioresource Technology 100 (2009) 5132–5139

major group of bacteria is shown in Fig. 3. A total of 48 unique Firmicutes (4.9%), Nitrospora (3.3%), Spirochaetes (3.3%), Bacteroides
OTUs were obtained out of which six chimeric OTUs were deleted (2.4%) and c-Proteobacteria (0.8%). Representative Proteobacterial
from the library. The rarefaction curve reached near to plateau and clones were used to construct a phylogenetic tree (Fig. 4). Several
Chaos value of 46.35 which is closer to the total number of clones bacterial clones found in the chocolate wastewater based MFC an-
indicating that the library has almost covered every possible ode chamber had shown close affiliation with the species reported
species present in the MFC anode chamber. The clone sequence earlier in different MFCs such as Thauera mechernichensis (Kim
analysis had shown wide diversity of bacterial groups in MFC et al., 2004, 2007), Azoarcus evansii KB740 (Kim et al., 2004; Phung
anode chamber affiliated to different phyla previously detected et al., 2004), Dechloromonas hortensis (Choo et al., 2006), and
in different MFCs (Tables 2 and 3). In terms of absolute numbers several genera such as Leptothrix sp., Legionella sp. (Phung et al.,
b-Proteobacteria constituted the most abundant division with 2004), Rhizobium sp., Treponema sp. (Ishii et al., 2008), Desulfomonile sp.
50.6% clones, followed by unclassified other bacteria (9.9%), a-Prote- (Aelterman et al., 2008), Burkholderia sp. (Ishii et al., 2008; Choo
obacteria (9.1%), other Proteobacteria (9%), Planctomycetes (5.8%), et al., 2006), Decholoromonas sp. (Kim et al., 2007), Planctomyces

Table 2
Summary of proteobacterial phylotypes retrieved from an MFC with activated sludge enriched with chocolate industry wastewater.

Name of Closest relative (NCBI Accession) % homology Closest cultivable species (NCBI Accession) % Class
OTUa homology
MFC_15G2 Leptothrix discophora SS-1 (L33975) 93 Leptothrix discophora SS-1 (L33975) 93 Betaproteobacteria
MFC_16G9 Uncultured beta proteobacterium clone: 187 (AB25290) 99 Aquincola tertiaricarbonis L10 (DQ656489) 98 Betaproteobacteria
MFC_17A6 Uncultured bacterium clone BD2 (FJ472352) 98 Aquincola tertiaricarbonis L10 (DQ656489) 98 Betaproteobacteria
MFC_04F8 Uncultured bacterium clone 35-10 (DQ833483) 96 Luteimonas aquatica RIB1-20 (EF626688) 94 Gammaproteobacteria
MFC_18B1 Uncultured bacterium clone SLB16 (DQ787671) 95 Pseudoxanthomonas sacheonensis BD-c54 (EF575564) Gammaproteobacteria
96
MFC_19D7 Bacterium SG-3 (AF548381) 98 Lysobacter brunescens KCTC 12130 (AB161360) 99 Gammaproteobacteria
MFC_20H11 Uncultured Xanthomonadaceae bacterium clone Plot 21-2C06 (EU193114) Dokdonella koreensis DS-123 09 (AY987368) 97 Gammaproteobacteria
96
MFC_21E8 Uncultured bacterium clone FHINOA29/1-7682 (FJ430185) 98 Pseudoxanthomonas yeongjuensis GR12-1 Gammaproteobacteria
(DQ438977) 89
MFC_22 Legionella-like amoebal pathogen HT99 (AY741401) 97 Methylococcus capsulatus ATCC 19069 (AY741401) 90 Gammaproteobacteria
MFC_25G12 Uncultured delta proteobacterium clone ST11-13(DQ501327) 97 Desulfomonile tiedjei ATCC 49306 (M26635) 85 Deltaproteobacteria
MFC_43D3 Uncultured Xanthomonadaceae bacterium (EF020325) 97) Lysobacter brunescens KCTC 12130 (AB161360) 93 Gammaproteobacteria
MFC_44F12 Uncultured beta proteobacterium clone AKYG827 (AY921721) 98 Burkholderia kururiensis JCM 10599 (AB024310) 90 Betaproteobacteria
MFC_45FA6 Thauera sp. Q4 (EU850616) 99 Thauera mechernichensis TL1 (Y17590) 97 Betaproteobacteria
MFC_46F10 Uncultured bacterium H30 (AF072924) 99 Azoarcus evansii KB740 (X77679) 96 Betaproteobacteria
MFC_47E7 Uncultured bacterium H30 (AF072924) 99 Dechloromonas hortensis MA-1 (AY277621) 96 Betaproteobacteria
MFC_48E10 Dechloromonas hortensis strain MA-1 (AY277621) 99 Dechloromonas hortensis strain MA-1 (AY277621) 99 Betaproteobacteria
MFC_49G6 Uncultured bacterium clone N1903_79 (EU104335) 98 Aquincola tertiaricarbonis L10 (DQ656489) 95 Betaproteobacteria
MFC_50G7 Uncultured bacterium clone N1903_79 (EU104335) 98 Aquincola tertiaricarbonis L10 (DQ656489) 95 Betaproteobacteria
MFC_51D2 Uncultured bacterium clone 69-12 (DQ833497) 96 Haliangium ochraceum SMP-2 (AB016470) 90 Deltaproteobacteria
MFC_26H7 Uncultured bacterium MERTZ_2CM_108 (AF424325) 91 Rhodanobacter lindaniclasticus RP5557 (AF039167) Gammaproteobacteria
82
MFC_29H8 Uncultured bacterium clone TUM-dMbac-MR1-B1-K1A-128 (FJ234907) 94 Acidithiobacillus thiooxidans ATCC 19377 (AY552087) Gammaproteobacteria
81
MFC_42D11 Uncultured bacterium (AB267045) 98 Thioalkalivibrio denitrificans ALJD (AF126545) 86 Gammaproteobacteria
MFC_23G6 Uncultured bacterium clone KGB200711-149 (EU881287) 98 Pelobacter acetylenicus WoAcy1 DSM2348 (X70955) Deltaproteobacteria
87
a
Only single clone is represented in the table.

Table 3
Summary of phylotypes other than proteobacteria retrieved from an MFC with activated sludge enriched with chocolate industry wastewater.

Name of OTUa Closest relative (NCBI Accession) % homology Closest cultivable species (NCBI Accession) % homology Class
MFC_27A12 Uncultured bacterium (AB286421) 94 Treponema medium G7201 (D85437) 81 Spirochaetes
MFC_28E8 Uncultured bacterium clone OTU_11 (EU083489) 91 Rhodopirellula baltica SH 1(BX294149) 82 Planctomycetes
MFC_29H6 Uncultured bacterium clone KIST-JJY107 (FJ232632) 99 Planctomyces maris DSM 8797 (AJ231184) 89 Planctomycetes
MFC_30A10 Uncultured bacterium mle1-48 (AF280867) 96 Caldilinea aerophila STL-6-O1 (AB067647) 83 Chloroflexi
MFC_31C1 Uncultured bacterium clone 5-53 (DQ833478) 88 Anaerolinea thermophila UNI-1 (AB046413) 81 Chloroflexi
MFC_32E10 Nitrospira defluvii (DQ059545) 99 Nitrospira defluvii (DQ059545) (99) Nitrospirales
MFC_33D12 Uncultured bacterium clone A4 (AY540495) 96 Prolixibacter bellariivorans F2(AY918928) 86 Bacteroidetes
MFC_34H3 Uncultured Gemmatimonadetes bacterium isolate BF23 (DQ839330) 98 Gemmatimonas aurantiaca T-27 (AB072735) 91 Gemmatimonadales
MFC_35C1 Uncultured bacterium clone B83 (AF407728) 97 Planctomyces maris DSM 8797(AJ231184) 85 Planctomycetes
MFC_36C1 Uncultured planctomycete clone MVS-107 (DQ676396) 93 Uncultured planctomycete clone MVS-107 (DQ676396) 93 Planctomycetes
MFC_37C1 Uncultured bacterium clone KIST-JJY074 (FJ208823) 96 Thermodesulfovibrio islandicus R1ha3X96726 80 Nitrospirales
MFC_38B8 Uncultured bacterium clone KIST-JJY074 (FJ208823) 96 Thermodesulfovibrio aggregans TGE-P1 (AB021302) 79 Nitrospirales
MFC_39A5 Uncultured bacterium isolate BM47 (EF126214) 88 Rhodopirellula baltica SH 1B (X294149) 80 Planctomycetes
MFC_40F11 Uncultured bacterium clone XJ109 (EF648155) 97 Spirochaeta stenostrepta ATCC 25083 (M88724) 89 Spirochaetes
MFC_41F6 Uncultured bacterium (AB266940) 99 Spirochaeta caldaria H1 (M71240) 86 Spirochaetes
MFC_52C6 Nitrospira cf. moscoviensis SBR1024 (AF155153) 99 Nitrospira cf. moscoviensis SBR1024 (AF155153) 99 Nitrospirales
MFC_53A9 Uncultured bacterium clone C1 (EF583494) 99 Dehalobacter restrictus PER-K23 (U84497) 88 Firmicutes
MFC_14F3 Uncultured bacterium clone AR-33 (DQ296474) 100 Prolixibacter bellariivorans F2(AY918928) 86 Bacteroidetes
a
Only single clone is represented in the table.
 S.A. Patil et al. / Bioresource Technology 100 (2009) 5132–5139 5137

Fig. 4. Phylogenetic tree showing the relationship among bacteria based on proteobacterial 16S rRNA gene sequences recovered from activated sludge and chocolate waste
based MFC anode (bold face) with reference sequences obtained through BLAST analysis. GenBank accession numbers are given in brackets. Bootstrap values (n = 1000
replicates) are reported as percentages. The scale bar represents substitution per site.

sp. (Kim et al., 2004), Thermodesulfovibrio sp. (Jong et al., 2006), Acidithiobacillus thiooxidans, Caldilinea aerophila, Anaerolinea
Spirochaetes (Jung and Regan, 2007), Nitrospora sp. (Ishii et al., thermophila, Nitrospira defluvii, Gemmatimonas aurantiaca,
2008), and Pelobacter sp. (Holmes et al., 2004). Burkholderia sp. Thioalkalivibrio denitrificans and Dehalobacter restrictus were not
has been reported in hydrogen production based on sequence reported as exoelectrogens so far as to our knowledge.
analysis information (Kalia and Purohit, 2008) and Prolixibacter Aquincola tertiaricarbonis, Lysobacter brunescens, Desulfomonile
bellariivorans, a sugar-fermenting anaerobe was found in tiedjei, Rhodanobacter lindaniclasticus, Pelobacter acetylenicus,
marine-sediment fuel cell (Holmes et al., 2007), which are pre- Rhodopirellula baltica, Dehalobacter restrictus, etc. have the ability
dicted to be involved in electricity generation. Many clone to degrade complex carbohydrates and other compounds. These
sequences found in the present studies showing close association microbes might have played role in degrading chocolate wastewa-
with different species such as Aquincola tertiaricarbonis, Luteimonas ter ingredients into simpler compounds, making easier for further
aquatica, Pseudoxanthomonas sacheonensis, Lysobacter brunescens, utilization by exoelectrogens. Some of the identified bacteria like
Dokdonella koreensis, Pseudoxanthomonas yeongjuensis, Haliangium Desulfomonile tiedjei, Candidatus Nitrospira defluvii are generally
ochraceum, Rhodanobacter lindaniclasticus, Rhodopirellula baltica, found in sewage sludge, which are not reported in MFCs to our
5138 S.A. Patil et al. / Bioresource Technology 100 (2009) 5132–5139

knowledge. Methylococcus capsulatus is methane utilizing microor- 4. Conclusions

ganism, which was possibly grown by using methane produced in
the anode chamber. However, methane production in anode is one The two-chambered MFC with chocolate industry wastewater
of the several limiting factors in electricity generation. This bacte- and activated sludge demonstrated its effectiveness for electricity
rium might be involved in reducing the methane concentration in generation. The study showed that the use of chocolate industry
the reactor. Leptothrix discophora SP-6, a type of manganese (Mn)- wastewater as both anolyte and catholyte for the electricity gener-
oxidizing bacteria, is known to be involved in microbiologically ation is promising, although, further studies are needed to opti-
influenced corrosion by increasing the electrochemical potential mize the process. Performance of the MFC also indicated its
of steel and other metals. This bacterium was found to modify efficiency in treatment of wastewater. Phylogenetic analysis of an-
the surface of glassy carbon in aqueous solution and increase its ode microbial community revealed the prominent presence of
potential and proposed to have potential as a catalyst in next gen- a-, b- and c-Proteobacteria, Firmicutes, Planctomycetes, Firmicutes,
eration MFCs (Nguyen et al., 2007). Acidithiobacillus thiooxidans has Nitrospora, Spirochaetes, Bacteroides and major population of
the ability to remove hydrogen sulfide from reactor (Aroca et al., unclassified bacteria. Various new bacterial groups were found in
2007), which might be involved in the H2S utilization in the anode. the anodic community indicating the diversity of microbes devel-
Some thermophiles like Anaerolinea thermophila, Thermodesulfovibrio oped in anode. The roles of these bacteria need to be explored to
islandicus, Thermodesulfovibrio aggregans and sulfur-oxidizing bacte- get clear idea about the microbes involved in anodic electron trans-
ria like Thioalkalivibrio denitrificans were also found in the anode fer. Overall, the finding suggests that the chocolate wastewater has
community adding to the microbial diversity of sewage sludge and a potential for future MFC practical applications as it can provide a
chocolate industry wastewater based MFC. It would be more readily biodegradable waste source for electricity generation.
speculative to describe the exact roles of most of the identified
bacteria based on only 16S rDNA sequence analysis. It is proposed
that some of these bacteria are not directly involved in electricity References
generation, but they might be involved in degrading the chocolate
American Public Health Association, American Water Works Association, Water
wastewater into simpler products which are further utilized by Pollution Control Federation, 1995. Standard Methods for the Examination of
exoelectrogens. The present architecture of the MFC, being batch Water and Wastewater, 19th ed. American Public Health Association,
reactor, can accommodate the bacterial DNA though it is dead or Washington, DC.
Aelterman, P., Versichele, M., Marzorati, M., Boon, N., Verstraete, W., 2008. Loading
not contributing for electron release. Hence, it should be proved
rate and external resistance control the electricity generation of microbial fuel
metabolically to confirm their role as exoelectrogens. Nevertheless, cells with different three-dimensional anodes. Bioresour. Technol. 99, 8895–
the role of electricity generation by these representatives cannot 8902.
be ruled out since it is not proved, yet. The majority of proteobacte- Aroca, G., Urrutia, H., Nunez, D., Oyarzun, P., Arancibia, A., Guerrero, K., 2007.
Comparison on the removal of hydrogen sulfide in biotrickling filters inoculated
ria enriched in the MFCs were different from the proteobacteria with Thiobacillus thioparus and Acidithiobacillus thiooxidans. Electr. J. Biotech. 10
reported in various earlier studies (Kim et al., 2007; Jung and Regan, (4), 514–520.
2007; Phung et al., 2004). In spite of the wide bacterial diversity in Ashelford, K.E., Chuzhanova, N.A., Fry, J.C., Jones, A.J., Weightman, A.J., 2006. New
screening software shows that most recent large 16S rRNA gene clone libraries
the activated sludge and chocolate industry wastewater based contain chimeras. Appl. Environ. Microbiol. 72, 5734–5741.
MFC, Shewenella sp. and Geobacter sp., the most prominent species Catal, T., Li, K., Bermek, H., Liu, H., 2008. Electricity production from twelve
reported in various MFCs, were not observed in our study. However, monosaccharides using microbial fuel cells. J. Power Sources 175, 196–200.
Choo, Y.F., Lee, J., Chang, I.S., Kim, B.H., 2006. Bacterial communities in microbial
the dominance of these organisms has not been found to be fuel cells enriched with high concentrations of glucose and glutamate. J.
obligatory to support high electrical power outputs using mixed Microbiol. Biotechnol. 16 (9), 1481–1484.
microbial communities (Rabaey et al., 2004). In this study, anode Felsenstein, J., 1993. PHYLIP (Phylogenetic Inference Package), Version 3.61.
University of Washington, Seattle, USA.
showed major proportion of Aquincola tertiaricarbonis L10 [4 OTUs], Ha, P.T., Tae, B., Chang, I.S., 2008. Performance and bacterial consortium of microbial
Lysobacter brunescens KCTC [2 OTUs], Dechloromonas hortensis [2 fuel cell fed with formate. Energy Fuels 22, 164–168.
OTUs], Planctomyces maris [2 OTUs], Prolixibacter bellariivorans [2 He, Z., Angenent, L.T., 2006. Application of bacterial biocathodes in microbial fuel
cells. Electroanalysis 18, 2009–2015.
OTUs], Rhodopirellula baltica [2 OTUs] and can be considered as most
Holmes, D.E., Bond, D.R., O’Neil, R.A., Reimers, C.E., Tender, L.R., Lovley, D.R., 2004.
feasible exoelectrogens in activated sludge and chocolate industry Microbial communities associated with electrodes harvesting electricity from a
wastewater based MFC. Also, the bacterial diversity of the activated variety of aquatic sediments. Microb. Ecol. 48, 178–190.
sludge and chocolate wastewater enriched MFC is much larger com- Holmes, D.E., Nevin, K.P., Woodard, T.L., Peacock, A.D., Lovley, D.R., 2007.
Prolixibacter bellariivorans gen. nov., sp. nov., a sugar-fermenting,
pared to the earlier reports. Different microbial samples can be used psychrotolerant anaerobe of the phylum Bacteroidetes, isolated from a
to enrich electron transferring microorganisms in MFCs. The use of marine-sediment fuel cell. Int. J. Syst. Evol. Microbiol. 57, 701–707.
activated sludge (Lee et al., 2003), anaerobic sludge (Rabaey et al., Huber, T., Faulkner, G., Hugenholtz, P., 2004. Bellerophon: a program to detect
chimeric sequences in multiple sequence alignments. Bioinformatics 20, 2317–
2004), marine sediment (Holmes et al., 2004) and domestic 2319.
wastewater (Liu et al., 2004) have been reported as a microbial Ishii, S., Hotta, Y., Watanabe, K., 2008. Methanogenesis versus electrogenesis:
inocula in anode of MFC. Although the MFC is an appropriate device morphological and phylogenetic comparisons of microbial communities. Biosci.
Biotechnol. Biochem. 72 (2), 286–294.
to enrich electricity producing microbial communities, a typical Jong, B.C., Kim, B.H., Chang, I.S., Liew, P.W., Choo, Y.F., Kang, G.S., 2006. Enrichment,
electricity generating microbial community has not been established performance, and microbial diversity of a thermophilic mediatorless microbial
yet (Aelterman et al., 2008). As per previously reported community fuel cell. Environ. Sci. Technol. 40, 6449–6454.
Jukes, T.H., Cantor, C.R., 1969. Evolution of protein molecules. In: Munro, H.N. (Ed.),
analysis data, there is no single emergent microorganism in the
Mammalian Protein Metabolism. Academic Press, New York, pp. 21–123.
bacterial communities that develops in the anode. Diverse bacterial Jung, S., Regan, J.M., 2007. Comparison of anode bacterial communities and
populations identified in MFCs with sludge and wastewater performance in microbial fuel cells with different electron donors. Appl.
Microbiol. Biotechnol. 77, 393–402.
show that electrochemical activity is not restricted to a few
Kalia, V.C., Purohit, H.J., 2008. Microbial diversity and genomics in aid of bioenergy.
phyla of bacteria. With a deeper understanding of these microbial J. Ind. Microbiol. Biotechnol. 35, 403–419.
communities, we could manipulate them to play an important Kim, B.H., Park, H.S., Kim, H.J., Kim, G.T., Chang, I.S., Lee, J., Phung, N.T., 2004.
role in MFCs in the future. The capability of growing more Enrichment of microbial community generating electricity using a fuel-cell-
type electrochemical cell. Appl. Microbiol. Biotechnol. 63, 672–681.
microorganisms within the anode compartment might result in Kim, J.R., Jung, S.H., Regan, J.M., Logan, B.E., 2007. Electricity generation and
the stable and effective operation of the MFCs and higher electricity microbial community analysis of alcohol powered microbial fuel cells.
output. Bioresour. Technol. 98, 2568–2577.
 S.A. Patil et al. / Bioresource Technology 100 (2009) 5132–5139 5139

Kimura, M., 1980. A simple method for estimating evolutionary rate of base Rabaey, K., Boon, N., Siciliano, S.D., Verhaege, M., Verstraete, W., 2004. Biofuel cells
substitution through comparative studies of nucleotide sequences. J. Mol. Evol. select for microbial consortia that self-mediate electron transfer. Appl. Environ.
16, 111–120. Microbiol. 70, 5373–5382.
Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L., Pace, N.R., 1985. Rapid Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory
determination of 16S ribosomal RNA sequences for phylogenetic analysis. Proc. Manual, second ed. Cold Spring Harbor, New York.
Natl. Acad. Sci. USA 82 (20), 6955–6959. Schloss, P.D., Handelsman, J., 2005. Introducing DOTUR, a computer program for
Lee, J., Phung, N.T., Chang, I.S., Kim, B.H., Sung, H.C., 2003. Use of acetate for defining operational taxonomic units and estimating species richness. Appl.
enrichment of electrochemically active microorganisms and their 16S rDNA Environ. Microbiol. 71, 1501–1506.
analyses. FEMS Microbiol. Lett. 223, 185–191. Schroeder, U., 2007. Anodic electron transfer mechanisms in microbial fuel cells and
Liu, H., Ramnarayanan, R., Logan, B.E., 2004. Production of electricity during their energy efficiency. Phys. Chem. Chem. Phys. 9, 2619–2629.
wastewater treatment using a single chamber microbial fuel cell. Environ. Sci. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary
Technol. 38, 2281–2285. Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599.
Liu, H., Logan, B.E., 2004. Electricity generation using an air-cathode single chamber Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the
microbial fuel cell in the presence and absence of a proton exchange membrane. sensitivity of progressive multiple sequence alignment through sequence
Environ. Sci. Technol. 38, 4040–4046. weighting, position-specific gap penalties and weight matrix choice. Nucleic
Liu, H., Grot, S., Logan, B.E., 2005. Electrochemically assisted microbial production of Acids Res. 22, 4673–4680.
electricity from acetate. Environ. Sci. Technol. 39, 4317–4320. Venkata Mohan, S., Sarvanan, R., Veer Raghuvulu, S., Mohankrishna, G., Sarma, P.N.,
Logan, B.E., Regan, J.M., 2006. Electricity-producing bacterial communities in 2008. Bioelectricity production from wastewater treatment in dual chambered
microbial fuel cells. Trends Microbiol. 14 (12), 512–518. microbial fuel cell (MFC) using selectively enriched mixed microflora: effect of
Logan, B.E., Hamelers, B., Rozendal, R., Schroeder, U., Keller, J., Freguia, S., Aelterman, catholyte. Bioresour. Technol. 99 (3), 596–603.
P., Verstraete, W., Rabaey, K., 2006. Microbial fuel cells: methodology and Wani, A.A., Suraksai, V.P., Siddharth, J., Raghavan, R.G., Patole, M.S., Ranade, D.,
technology. Environ. Sci. Technol. 40 (17), 5181–5192. Shouche, Y.S., 2006. Molecular analysis of microbial diversity associated with
Maidak, B.L., Cole, J.R., Parker, C.T., Garrity, G.M., Larsen, N., Li, B., Lilburn, T.G., the Lonar soda lake in India: an impact crater in basalt area. Res. Microbiol. 157,
McCaughey, M.J., Olsen, G.J., Overbeek, R., Pramanik, S., Schmidt, T.M., Tiedje, 928–937.
J.M., Woese, C.R., 1999. A new version of the RDP (Ribosomal Database Project). Xia, X., Xie, Z., 2001. DAMBE: software package for data analysis in molecular
Nucleic Acids Res. 27, 171–173. biology and evolution. J. Hered. 92, 371–373.
Nguyen, T.A., Lu, Y., Song, S., Shi, X., 2007. Carbon and steel surfaces modified by Yeates, C., Gillings, M.R., Davison, A.D., Altavilla, N., Veal, D.A., 1997. PCR
Leptothrix discophora SP-6: characterization and implications. Environ. Sci. amplification of crude microbial DNA extracted from soil. Lett. Appl.
Technol. 41 (23), 7987–7996. Microbiol. 25, 303–307.
Phung, N.T., Lee, J., Kang, K.H., Chang, I.S., Gadd, G.M., Kim, B.H., 2004. Analysis of Zehnder, A.J.B., Huser, B.A., Brock, T.D., Wuhrmann, K., 1980. Characterization of an
microbial diversity in oligotrophic microbial fuel cells using 16S rDNA acetate-decarboxylating, non-hydrogenoxidizing methane bacterium. Arch.
sequences. FEMS Microbiol. Lett. 233, 77–82. Microbiol. 124, 1–11.