J. Microbiol. Biotechnol. (2011), 21(2), 187–191doi: 10.4014/jmb.1008.08019First published online 27 November 2010

Characterization of Microbial Fuel Cells Enriched Using Cr(VI)-ContainingSludge

Ryu, Eun Yeon1†

, Mia Kim2†

, and Sang-Joon Lee2*

1BIO-IT Fusion Technology Research Institute, Pusan National University, Busan 609-735, Korea 2Department of Microbiology, Pusan National University, Busan 609-735, Korea

Received: August 18, 2010 / Revised: November 9, 2010 / Accepted: Novemer 10, 2010

Microbial fuel cells (MFCs) were successfully enriched

using sludge contaminated with Cr(VI) and their

characteristics were investigated. After enrichment, the

charge of the final 10 peaks was 0.51 C ± 1.16%, and the

anodic electrode was found to be covered with a biofilm.

The enriched MFCs removed 93% of 5 mg/l Cr(VI) and

61% of 25 mg/l Cr(VI). 16S rDNA DGGE profiles from

the anodic electrode indicated that β-Proteobacteria,

Actinobacteria, and Acinetobacter sp. dominated. This

study is the first to report that electrochemically active

and Cr(VI)-reducing bacteria could be enriched in the

anode compartment of MFCs using Cr(VI)-containing

sludge and demonstrates the Cr(VI) removal capability of

such MFCs.

Keywords: Microbial fuel cell, hexavalent chromium, Cr(VI)

reduction, microbial diversity, denaturing gradient gel

electrophoresis (DGGE), electrochemically active bacteria

Hexavalent chromium [Cr(VI)] is a critical pollutant that is

a mainstay of a wide variety of industries. Effluents from

these industries contain Cr(VI) at concentrations ranging

from tens to hundreds of milligram per liter [3]. Dissolved

Cr(VI) transverses cell membranes, posing a serious risk as

a carcinogen and mutagen [4, 7]. Thus, removal of Cr(VI)

from industrial wastewater is an important goal to pursue.

Cr(VI) removal is traditionally accomplished by one of

various methods [1, 16, 20]. Physiological and chemical

approaches, using adsorbents, form the basis of conventional

techniques [16]. Biosorption has also been reported to be a

low-cost and effective method [23].

Microbial fuel cells (MFCs) can treat wastewater through

the catalytic activity of communities of electrochemically

active bacteria (EAB) [5]. Recently, several types of MFCs

have been developed for the simultaneous treatment of

wastewater and generation of electricity [9, 15].

Cr(VI) removal has been attempted using cathodic

reduction of MFCs [24]. However, there have been no

reports concerning removal of Cr(VI) by MFC bacteria in

the anode compartment using sludge containing Cr(VI) as

the inoculum.

In this study, we attempted to enrich EAB using Cr(VI)-

containing sludge, investigated Cr(VI) reduction activity,

and analyzed the bacterial community in the anode

compartment of the MFC.

MATERIALS AND METHODS

Microbial Fuel Cell System

Mediatorless MFCs were arranged (dimensions, 40×80×32 mm).

Graphite felts (GF series; Electrosynthesis, USA) and Nafion 424

(Dupont, USA) were used as electrodes and cation-exchange

membrane, respectively. Electrical signals were recorded with a

model 2700 Multimeter/Data Acquisition System (Keithley, USA).

The current and the charge in coulombs [current (I)×time (t)] were

calculated [11].

Inoculum and Enrichment Procedure

To cbtain an inoculum, sludge containing Cr(VI) was collected from

a leather tanning wastewater treatment plant located in Busan,

Korea. The anode compartment was fed synthetic wastewater [11]

and the cathode compartment was fed distilled water saturated with

oxygen. The flow rate was 1.2 ml/min for 15 min every hour. The

temperature of the MFC was maintained at 35oC.

PCR-DGGE and Sequencing of 16S rDNA

DNA from the anodic electrodes was extracted using the Fast DNA

SPIN for soil kit (MP Biomedicals, USA). 16S rDNA was amplified

by the polymerase chain reaction (PCR) using the universal primer

pair 27F/1492R, and re-amplified using the primer pair 341F-GC/

518R [12]. Amplicons were separated on an 8% polyacrylamide gel.

After denaturing gradient gel electrophoresis (DGGE), bands were

*Corresponding authorPhone: +82 (51) 510 2268; Fax: +82 (51) 514 1778;E-mail: sangjoon@pusan.ac.kr†The first two authors contributed equally to this work.

188 Ryu et al.

excised and re-amplified using the primer set 341F/518R. PCR

products were cloned and sequenced using the T-Blunt Vector

(Solgent, Korea). All sequences were aligned using CLUSTAL W

software version 1.7 [22].

Microscopy

The surfaces of the anodic electrodes were examined by scanning

electron microscopy (SEM). Samples were examined in a S3500N

instrument (Hitachi, Japan) operating at 20 kV. Samples were also

examined by confocal laser scanning micrography (CLSM) using a

CLSM 510 apparatus (Zeiss, Germany).

Cr(VI) Reduction Test

Potassium chromate (K2CrO4; Junsei Chemical, Japan) was added to

synthetic wastewater. The Cr(VI) concentrations in the effluents was

measured using the 1,5-diphenylcarbazide method [2].

RESULTS AND DISCUSSION

Enrichment of Bacterial Consortium in MFCs Using

Cr(VI)-Containing Sludge

EAB were enriched in MFCs using Cr(VI)-containing

sludge [0.43 mg/l Cr(VI)]. The resulting current profile

was compared with that from general enrichment using

activated sludge from a sewage treatment plant (Fig. 1).

Immediately following the addition of either type of

sludge, the MFC potential increased up to 0.5-0.6 V.

Under 200 Ω of load, the potential of the MFC inoculated

with sewage sludge decreased to 7.3×10-5 V (Fig. 1A),

whereas that of the MFC inoculated with Cr(VI)-containing

sludge was 8.0×10-3 V (Fig. 1B). The concentrations of

sulfide and chloride in the Cr(VI)-containing sludge were

1.2×103 mg/l and 2.2×103 mg/l, respectively. The high

anion content likely caused the relative high potential at

the initial stage [8]. The enrichment of MFCs using Cr(VI)-

containing sludge and sewage sludge was completed in 6

and 8 days, respectively.

The anodic electrodes were observed using SEM and

CLSM. Numerous twisted carbon fiber strips produced

graphite felts (Fig. 2A). After enrichment, the electrode

Fig. 1. Current profile during the enrichment procedure fromMFCs inoculated with (A) activated sludge from a sewagetreatment plant and (B) Cr(VI)-containing sludge from a leathertanning wastewater treatment plant.

Fig. 2. SEM images of the (A) electrode before enrichment and(B and C) electrodes of the anode compartment of MFC enrichedusing Cr(VI)-containing sludge. The scale bars represent dimensions as shown.

MFC ENRICHED USING SLUDGE CONTAMINATED BY CR(VI) 189

was found to be covered with bacteria that were mainly

rod-shaped, 1.0-2.5 µm long, and 0.4-0.8 µm wide (Fig. 2B

and 2C). A representative CLSM image shows the bacterial

clumps and biofilm that were evident along the length of

the electrode (Fig. 3).

Cr(VI) Reduction by MFCs

After the enrichment procedure, the Cr(VI) removal rate

was analyzed. As shown in Table 1, 93% of 5 mg/l Cr(VI)

and 61% of 25 mg/l Cr(VI) were removed by the MFCs.

The Cr(VI) removal effect by the carbon felt electrode alone

was also determined, and was found to be about 20%.

Thus, the Cr(VI) removal rate attributable to microbial

factors in the MFCs was 68% of 5 mg/l Cr(VI) and 42% of

25 mg/l Cr(VI). MFC electrical signals were also affected

by the presence of Cr(VI). Although decreases in the MFC

current were observed during injection of Cr(VI)-containing

synthetic wastewater, when injection of Cr(VI)-free synthetic

wastewater was initiated, the current was recovered, up to

about 90% in the case of both Cr(VI) concentrations,

compared with the current before the addition of Cr(VI)

(Fig. 4). MFC-mediated Cr(VI) removal can be explained

by physical and biological phenomena. Firstly, Cr(VI)

could be removed by physical adsorption on the electrode

[18]; the carbon felt consists of a network and possesses a

high surface area [6]. Secondly, Cr(VI) could be removed

by a biological reaction such as biosorption or reduction

to Cr(III) [13, 21]. A previous study reported that the

concentration of Cr(VI) was reduced from 5 mg/l to 1 mg/l

in 3 days by Shewanella oneidensis, which is known to be

an EAB [10, 13]. It seems that such bacteria can reduce

Cr(VI) to Cr(III) using the former as an electron acceptor,

thus causing a decrease in current.

Fig. 3. CSLM image of the anodic electrode of MFC enrichedusing Cr(VI)-containing sludge.

Fig. 4. Current patterns from MFCs injected with syntheticwastewater containing (A) 5 mg/l Cr(VI) and (B) 25 mg/l Cr(VI).

Table 1. Analysis of charge and Cr(VI) removal rate by MFCs.

Cr(VI) conc. of influent

5 mg/l 25 mg/l

Charge (coulombs)aBefore Cr(VI) injection 0.37±0.003 0.17±0.023

During Cr(VI) injection 0.32±0.011 0.08±0.019

After Cr(VI) injection 0.33±0.008 0.10±0.015

Cr(VI) removal rate (%)MFC inoculated with sludge 93 61

Uninoculated MFC 25 19a

Average values of nine peaks under each condition. The peaks produced when switching the medium were excluded from the calculation.

Removal rates were calculated by measuring the Cr(VI) concentration in MFC effluents.

190 Ryu et al.

Bacterial Community Analysis by PCR-DGGE of 16S

rDNA

The bacterial community in the MFC was analyzed by

PCR-DGGE. Fig. 5 shows the bacterial profiles of the

inoculum and the anodic electrode after the enrichment

procedure. DGGE revealed similar patterns for the inoculum

and electrode. However, the main bacterial groups had

changed. Among those species found after enrichment,

Clostridium sp. (EB3) is a Cr(VI) reducer [14] and has

been identified as an EAB from mediatorless MFCs [19].

Acinetobacter sp. is also a Cr(VI) reducer, but it does not

have electrochemical activity [21]; it is a strict aerobic

bacterium, capable of growing in the anode compartment

using oxygen diffused through the cation-exchange membrane

from the cathode [15]. Cr(VI) reduction processes by

Cr(VI)-reducing bacteria (CRB) require protons [17, 25],

which might be supplied by the metabolism of fermentative

bacteria [5]. Based on the experimental results, it is

presumed that the microbial consortium, composed of

CRB with/without electrochemical activity and non-CRB

with/without electrochemical activity, was formed in the

anode compartment. The electricity from the MFC was

generated by EAB with/without Cr(VI) reduction in the

absence of Cr(VI). When Cr(VI) was present, it was used

as an electron acceptor by CRB with electrochemical

activity instead of interacting with the electrode, thus

causing a decrease in current. Moreover, Cr(VI) reduction

was likely performed by CRB without electrochemical

activity such as Acinetobacter, and fermentative bacteria

could have supplied the protons for Cr(VI) reduction.

Although a more detailed study of the interactions between

members of the bacteria consortium and the mechanisms

of Cr(VI) reduction is required, this is the first attempt to

enrich microbes using Cr(VI)-containing sludge, to observe

Cr(VI) removal, and to analyze the microbial community

in the anode.

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