9
Using sediment microbial fuel cells (SMFCs) for bioremediation of polycyclic aromatic hydrocarbons (PAHs) Mohammad Sherafatmand, How Yong Ng Center for Water Research, Department of Civil and Environmental Engineering, National University of Singapore, Block E1A #07-03, 1 Engineering Drive 2, Singapore 117576, Singapore highlights PAHs bioremediation using a microbial fuel cell. PAHs bioremediation in aerobic or anaerobic environment of cathodic chamber. A consistent power density output for more than two months. Smaller internal resistance compared to literatures. A prospective method for in situ bioremediation. article info Article history: Received 22 April 2015 Received in revised form 31 May 2015 Accepted 1 June 2015 Available online xxxx Keywords: Sediment microbial fuel cell (SMFC) Polycyclic aromatic hydrocarbons (PAHs) Bioremediation Contaminated soil Power generation abstract In this study, a sediment microbial fuel cell (SMFC) was explored to bioremediate polycyclic aromatic hydrocarbons (PAHs) in water originated from soil. The results showed consistent power generations of 6.02 ± 0.34 and 3.63 ± 0.37 mW/m 2 under an external resistance of 1500 O by the aerobic and anaer- obic SMFC, respectively. Although the power generations were low, they had relatively low internal resis- tances (i.e., 436.6 ± 69.4 and 522.1 ± 1.8 O for the aerobic and anaerobic SMFC, respectively) in comparison with the literature. Nevertheless, the significant benefit of this system was its bioremediation capabilities, achieving 41.7%, 31.4% and 36.2% removal of naphthalene, acenaphthene and phenanthrene, respectively, in the aerobic environment and 76.9%, 52.5% and 36.8%, respectively, in the anaerobic envi- ronment. These results demonstrated the ability of SMFCs in stimulating microorganisms for bioremedi- ation of complex and recalcitrant PAHs. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compound that consists of two or more fused benzene rings and/or pentacyclic molecules that are arranged in various structural con- figurations. They are highly recalcitrant molecules that can persist in the environment due to their hydrophobicity and low water sol- ubility (Bamforth and Singleton, 2005; Cerniglia, 1992). PAHs are ubiquitous in the environment, and originate from either natural or anthropogenic sources (Bamforth and Singleton, 2005). PAHs are one of the most prevalent contaminates found in soil (Bamforth and Singleton, 2005; Liebega and Cutright, 1999). The origin of PAH contaminated soils include anthropogenic sources such as abandoned manufactured gas sites, leaking underground storage tanks, wood treatment sites and industrial processes (Liebega and Cutright, 1999). Natural processes can also provide a source of PAHs such as volcanic eruptions and forest fires (Blumer, 1976). For instance, they have been found in marine sed- iments such as San Diego Bay, California and the Central Pacific Ocean (Coates et al., 1997; Ohkouchi et al., 1999). Biostimulation & bioaugmentations are widely known tech- nologies to remediate hydrocarbons-polluted sites (Amezcua-Allieri et al., 2003, 2012; Singer et al., 2005). Biodegradation is occurred by breaking down of PAHs using microorganisms, either in the presence of oxygen (i.e., aerobic con- dition) or without oxygen (i.e., anaerobic condition). A variety of aerobic bacteria, fungi and enzymes have been specified as the spe- cies that can use PAHs as carbon and energy sources (Haritash and Kaushik, 2009). The limiting factors with aerobic biodegradation such as a very thin layer of oxic zone in the soil, aeration expenses and the tendency of PAHs to be accumulated in the soil rather than dissolving in water or suspending in air, show the significance and potential of anaerobic biodegradation (Liang et al., 2007). PAHs are http://dx.doi.org/10.1016/j.biortech.2015.06.002 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved. Corresponding author. Tel.: +65 6516 4777. E-mail address: [email protected] (H.Y. Ng). Bioresource Technology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment microbial fuel cells (SMFCs) for bioremediation of polycyclic aromatic hydro- carbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.06.002

Using sediment microbial fuel cells (SMFCs) for bioremediation of polycyclic aromatic hydrocarbons (PAHs)

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Page 1: Using sediment microbial fuel cells (SMFCs) for bioremediation of polycyclic aromatic hydrocarbons (PAHs)

Bioresource Technology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Using sediment microbial fuel cells (SMFCs) for bioremediationof polycyclic aromatic hydrocarbons (PAHs)

http://dx.doi.org/10.1016/j.biortech.2015.06.0020960-8524/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +65 6516 4777.E-mail address: [email protected] (H.Y. Ng).

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment microbial fuel cells (SMFCs) for bioremediation of polycyclic aromaticcarbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.06.002

Mohammad Sherafatmand, How Yong Ng ⇑Center for Water Research, Department of Civil and Environmental Engineering, National University of Singapore, Block E1A #07-03, 1 Engineering Drive 2, Singapore117576, Singapore

h i g h l i g h t s

� PAHs bioremediation using a microbial fuel cell.� PAHs bioremediation in aerobic or anaerobic environment of cathodic chamber.� A consistent power density output for more than two months.� Smaller internal resistance compared to literatures.� A prospective method for in situ bioremediation.

a r t i c l e i n f o

Article history:Received 22 April 2015Received in revised form 31 May 2015Accepted 1 June 2015Available online xxxx

Keywords:Sediment microbial fuel cell (SMFC)Polycyclic aromatic hydrocarbons (PAHs)BioremediationContaminated soilPower generation

a b s t r a c t

In this study, a sediment microbial fuel cell (SMFC) was explored to bioremediate polycyclic aromatichydrocarbons (PAHs) in water originated from soil. The results showed consistent power generationsof 6.02 ± 0.34 and 3.63 ± 0.37 mW/m2 under an external resistance of 1500 O by the aerobic and anaer-obic SMFC, respectively. Although the power generations were low, they had relatively low internal resis-tances (i.e., 436.6 ± 69.4 and 522.1 ± 1.8 O for the aerobic and anaerobic SMFC, respectively) incomparison with the literature. Nevertheless, the significant benefit of this system was its bioremediationcapabilities, achieving 41.7%, 31.4% and 36.2% removal of naphthalene, acenaphthene and phenanthrene,respectively, in the aerobic environment and 76.9%, 52.5% and 36.8%, respectively, in the anaerobic envi-ronment. These results demonstrated the ability of SMFCs in stimulating microorganisms for bioremedi-ation of complex and recalcitrant PAHs.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a class of organiccompound that consists of two or more fused benzene rings and/orpentacyclic molecules that are arranged in various structural con-figurations. They are highly recalcitrant molecules that can persistin the environment due to their hydrophobicity and low water sol-ubility (Bamforth and Singleton, 2005; Cerniglia, 1992). PAHs areubiquitous in the environment, and originate from either naturalor anthropogenic sources (Bamforth and Singleton, 2005).

PAHs are one of the most prevalent contaminates found in soil(Bamforth and Singleton, 2005; Liebega and Cutright, 1999). Theorigin of PAH contaminated soils include anthropogenic sourcessuch as abandoned manufactured gas sites, leaking undergroundstorage tanks, wood treatment sites and industrial processes

(Liebega and Cutright, 1999). Natural processes can also providea source of PAHs such as volcanic eruptions and forest fires(Blumer, 1976). For instance, they have been found in marine sed-iments such as San Diego Bay, California and the Central PacificOcean (Coates et al., 1997; Ohkouchi et al., 1999).

Biostimulation & bioaugmentations are widely known tech-nologies to remediate hydrocarbons-polluted sites(Amezcua-Allieri et al., 2003, 2012; Singer et al., 2005).Biodegradation is occurred by breaking down of PAHs usingmicroorganisms, either in the presence of oxygen (i.e., aerobic con-dition) or without oxygen (i.e., anaerobic condition). A variety ofaerobic bacteria, fungi and enzymes have been specified as the spe-cies that can use PAHs as carbon and energy sources (Haritash andKaushik, 2009). The limiting factors with aerobic biodegradationsuch as a very thin layer of oxic zone in the soil, aeration expensesand the tendency of PAHs to be accumulated in the soil rather thandissolving in water or suspending in air, show the significance andpotential of anaerobic biodegradation (Liang et al., 2007). PAHs are

hydro-

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2 M. Sherafatmand, H.Y. Ng / Bioresource Technology xxx (2015) xxx–xxx

a common contaminant in anaerobic environments such as soiland sediment (Coates et al., 1997; Coates, 1996; Genthner et al.,1997). Even anaerobic zones could be developed in aerobic envi-ronments by depletion of oxygen during aerobic respiration andnot replenishing at the same rate (Bedessem et al., 1997). This sug-gests that anaerobic zones could be easily established and thusanaerobic biodegradation would occur.

Anaerobic biodegradation of organic pollutants is an importantpathway in nature due to the ability of microorganisms in removalof organic compounds under anoxic conditions (Huang et al.,2011). Basically, anaerobic biodegradation needs an electronacceptor such as Fe(III) oxides, nitrate or sulfate. Due to the abun-dance of sulfate in the soil, especially marine environments, biore-mediation of PAHs in many instances would be most effectiveunder sulfate-reducing conditions (Chang et al., 2002; Kraig,2000). The possibility of oxidation of PAHs undersulfate-reducing conditions has been investigated by Coates(1996).

A microbial fuel cell (MFC) is a device that generates electricityby bacterial oxidation of substrates that are either organic or inor-ganic (Rezaei et al., 2007; Rabaey et al., 2006; Logan, 2008). Thiscan be achieved when bacteria switch from a natural electronacceptor such as oxygen or nitrate, to an insoluble acceptor suchas the MFC anode. This transfer can occur either viamembrane-associated compounds or soluble electron shuttles.The electrons then flow through an external resistor to a cathode,at which the electron acceptor is reduced (Rabaey and Verstraete,2005). In this process, which involves a wide range of microorgan-isms (Logan, 2009; Lovley, 2008), organic hydrocarbons would bedegraded at the anode of the MFC.

A sediment microbial fuel cell (SMFC) is a type of MFC that hasrecently attracted significant attentions (Rezaei et al., 2007; Huanget al., 2011) due to its unique property of removing organic com-pounds from the soil/sediment. SMFCs typically consist of an anodeburied in a reduced matrix (soil) and a cathode in the overlaying,oxidized water layer (Rezaei et al., 2007; Logan, 2008; Tenderet al., 2002). However, there is no detailed research into the abilityof MFC/SMFC for bioremediation of complex compounds such asPAHs. All the former studies have been done on non-complex com-pounds present in the soil/sediment except the studies on Chitin20 and Chitin 80 done by Rezaei et al. (2007) and anaerobicbiodegradation of diesel by Morris et al. (2009). This study investi-gated the ability of SMFC for bioremediation of PAHs in contami-nated soil under either aerobic or anaerobic environmentprovided in the cathodic chamber.

2. Materials

2.1. Sediment

Sediment (0–20 cm depth) and the lake water were collectedfrom the MacRitchie Reservoir (Singapore). They were placed intoclean polycarbonate jars and transported to the laboratory. All sed-iment were sieved through a 2-mm sieve to remove plant debrisand other terrestrial leaves and then homogenized by mixing witha stainless steel spatula prior to use.

2.2. Reagents

PAHs (naphthalene, acenaphthene and phenanthrene), tetrahy-drofuran (THF), methanol, 2-propanol, potassium nitrate andpotassium sulfate were purchased from Sigma Aldrich. All stan-dards and working solutions were stored at 4 �C. Deionized waterwas obtained from a Mili-Q water purification system (Milipore,

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment mcarbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech

USA). Bakerbond SPE columns C18 (2.5 g) for solid phase extractionwere purchased from Agilent Technologies.

2.3. Sample pretreatment (SPE method) – PAHs extraction

Since the samples were sediment, pretreatment of the samplesaccording to the method developed by Kootstra et al. (1995) wascarried out prior to analysis. The procedure of the pretreatmentwas as follows: a 10-g amount of soil was placed into a 50-ml tubewith 20 ml of acetone and the mixture was shaken for 30 min.After centrifugation at 1000 g for 5 min, exactly 10 ml of the mix-ture was then pipetted into a 100-ml volumetric flask togetherwith 5 ml of 2-propanol. The sample was brought to 100 ml withHPLC-grade water.

C18 were conditioned with 1 � 3 ml of methanol, followed bytwo times of 3 ml of water-2-propanol (9:1, v/v). The 100-ml sam-ple solution was loaded onto the SPE column under vacuum. Thenthe column was washed with 3 ml of methanol-water (50:50, v/v).The PAHs were eluted with two times of 1.5 ml of THF. The first1.5 ml has to soak the cartridge for two minutes before eluting.After elution, the final THF extract passed through a filter and thenwas injected into the GC/MS. All flows through the cartridge wereabout 2 ml/min.

2.4. Sample pretreatment-sediment characteristics

Sediment samples were analyzed for the amount of reducingcompounds present (i.e., sulfate, nitrate, phosphate, iron and man-ganese). Sulfate, phosphate, iron, manganese and other ions weremeasured by extracting from the sediment and analyzing with anIon Chromatograph (IC). Nitrate was measured using the TNTNitroVer Test Kit and DR5000 UV–vis spectrophotometer (Hach).All extracts were filtered through a 0.45-lm GA-8 membrane filterbefore being analyzed by the IC or Spectrophotometer.

2.5. Sediment MFCs (SMFCs)

2.5.1. Substrate2.5.1.1. PAHs. A solution of three selected PAHs (i.e., naphthalene,acenaphthene and phenanthrene) was added to the sediment tocreate contaminated sediment with PAHs for the study (50 ppmof each). First, PAHs were dissolved into THF (solvent) and thenadded to the sediment and mixed by a stainless steel spatula toget a homogenized mixture of sediment and PAHs.

2.5.1.2. Terminal electron acceptors (TEAs). For aerobic SMFC reac-tors, no external TEA was added since oxygen was introduced inthe cathodic chamber. However, for anaerobic SMFC reactors,nitrate and sulfate were added to provide potential TEAs for thereduction reaction. Nitrate and sulfate would be utilized for stim-ulating nitrate-reducing and sulfate-reducing bacteria, respec-tively, that have been known for the biodegradation of PAHs inthe soil in an anaerobic environment (Coates et al., 1997; Coates,1996; Meckenstock et al., 2000; Zhang et al., 2000).

2.5.1.3. Final substrate. The final substrates for aerobic and anaero-bic SMFCs consist of PAHs and the TEAs (i.e., nitrate and sulfateonly for anaerobic SMFC) were made by mixing PAH solution andTEA solution according to Table 1.

2.5.2. ElectrodesIsomolded graphite plates were used as the electrodes in this

experiment (Graphite Store Pte Ltd, USA). One graphite plate(10.16 cm � 10.16 cm � 0.318 cm) as the anode and one with thesame dimensions as the cathode (Fig. 1a and c) were used for eachSMFC reactor.

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Table 1The substrate used for the aerobic and anaerobic reactors.

Cathodeenvironment

PAHs TEA Concentration Reactor

Aerobic No Oxygen – AR2Aerobic Yes Oxygen – AR1, AR3 & AR4Anaerobic No Nitrate and sulfate 10 mM for each TEA AnR2Anaerobic Yes Nitrate and sulfate 10 mM for each TEA AnR1, AnR3 & AnR4

Fig. 1. Schematic diagram of the experimental setup. (a) Aerobic SMFC (AR2, AR3 and AR4), (b) aerobic non-SMFC (AR1), (c) anaerobic SMFC (AnR2, AnR3 and AnR4) and (d)anaerobic non-SMFC (AnR1).

M. Sherafatmand, H.Y. Ng / Bioresource Technology xxx (2015) xxx–xxx 3

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment microbial fuel cells (SMFCs) for bioremediation of polycyclic aromatic hydro-carbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.06.002

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2.5.3. SMFC construction and operationEight SMFC reactors (AR1 to AR4 and AnR1 to AnR4 under aer-

obic and anaerobic environments, respectively), made of Plexiglas,were operated simultaneously. The bottom of AR1, AR3, AR4, AnR1,AnR3 and AnR4 were filled with the prepared substrate as men-tioned earlier and is summarized in Table 1. AR1 and AnR1 werereactors comprised of only lake water on top of the sediment with-out any electrodes, and they were used to determine the back-ground bioremediation done by the indigenous microorganismsin a non-SMFC environment (denoted as non-SMFC reactors).AR3, AR4, AnR3 and AnR4 were the duplicated SMFC reactors inaerobic and anaerobic environments, respectively, and were con-structed by placing the electrodes in horizontal position in theheight of 4 cm (anode) and 15 cm (cathode) from the bottom andthen the lake water was added as the water column. AR2 andAnR2 were constructed similar as the duplicated SMFC reactors(i.e. AR3, AR4, AnR3 and AnR4), and the difference was that thesubstrate in it was the clean sediment (i.e., without PAHs) to mon-itor the background electricity generation. Fig. 1 shows the sche-matic of non-SMFC & SMFC reactors in both aerobic andanaerobic conditions and Table 2 displays a summary of the con-structed reactors. The circuit was completed using a 1500-O resis-tor for each cell. Voltages for all reactors were monitored across theresistance every 30 min using a data acquisition system. For aero-bic SMFC reactors (i.e., AR1 to AR4), air was introduced by a finebubble diffuser suspended in the overlaying water near the cath-odes in order to maintain oxic condition (i.e., dissolved oxygenconcentration of 3-4 mg/L) in the cathode chamber (Hong et al.,2010). For anaerobic mode, all the SMFC reactors (i.e., AnR1 toAnR4) were completely closed using rubbers and caps to maintainanaerobic condition inside the reactors. All the SMFC reactors wereoperated at ambient temperature (�27 �C) and the water loss dueto evaporation was compensated every two days by topping upwith tap water.

2.6. Measurement

2.6.1. Cell voltagesCell voltages (V) were measured using a data acquisition system

(ARRAY M3500A, Array Electronic Co., Ltd.) connected to acomputer.

2.6.2. Electrode referenceAnode and cathodes potentials were measured by an Ag/AgCl

reference electrode (Metrohm Pte Ltd).

2.6.3. Current/powerCurrent (I) was calculated using the ohm’s law (R = V/I), where R

is the external resistance (O) and V is the measured cell Voltage (V).Power (P) was calculated as P = VI and normalized by the anodesurface area. The maximum power density was measured by vary-ing the external resistance from 50 kO to 25 O with 15 min interval

Table 2A summary of the SMFC and non-SMFC reactors.

Type Reactor SMFC Contaminatedsediment

Initial PAHsconc.

External TEAs(nitrate & sulfate)

Aerobic AR1 No Yes 50 ppm (each) NoAerobic AR2 Yes No – NoAerobic AR3 Yes Yes 50 ppm (each) NoAerobic AR4 Yes Yes 50 ppm (each) NoAnaerobic AnR1 No Yes 50 ppm (each) YESAnaerobic AnR2 Yes No – YESAnaerobic AnR3 Yes Yes 50 ppm (each) YESAnaerobic AnR4 Yes Yes 50 ppm (each) YES

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment mcarbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech

time for allowing the voltage to be stabilized. The internal resis-tances of the SMFC reactors were calculated using the slope ofpolarization curve as reported by Logan (2008).

2.6.4. Gas chromatography/mass spectrometry (GC/MS)A Shimadzu 2010 GC system with a mass spectrometer detector

(MS) were used for the determination of PAH concentration. Thefollowing analytical conditions were used: capillary column usedwas the DB-5 MS (30 m, 0.25 mm, 0.25 lm film thickness); thepressure was 44.2 kPa; the injector temperature was maintainedat 300 �C, the initial temperature used was 70 �C and was heldfor 2 min; the temperature was then increased at a rate of15 �C/min until it reached the first isotherm of 200 �C and was heldfor 4 min; the temperature was further increased a rate of 5 �C/minto reach the second isotherm at 300 �C and held for 5 min. Ionsource and interface temperatures were maintained at 250 �Cand the peaks captured by the SIM method. PAHs were identifiedby retention times and characteristic ions of identified compounds.

2.6.5. Ion chromatography (IC)A Dionex DX500 ion chromatograph (Dionex Corporation,

Sunnyvale, CA, USA), equipped with a GP50 gradient pump, aLC25 chromatography oven and an AD20 absorbance detectorwas used for the detection of ions in the samples. The analyticalcolumn used was Thermo Scientific, DIONEX ASRS™ 300 (4 mm).

2.6.6. UV–vis spectrophotometerA DR 5000 UV–vis spectrophotometer (HACH, Colorado, USA)

was used for measuring the nitrate concentration. The kit usedwas the TNT NitraVer � 50 tests, high range (0–30 mg/l NO3

�–N).

2.6.7. Total organic carbon (TOC)Total organic carbon (TOC) of water and sediment samples were

analyzed using a Total Carbon Analyzer (Shimadzu TOC-L) and aSolid Sample Module (SSM) associated with TOC-V, respectively.

2.6.8. Cyclic voltammetry (CV)Bio-electrochemical behavior of mixed communities in the

reactors was studied by cyclic voltammetry (CV) using a poten-tiostat system (Metrohm, Autolab). All the assays were performedby considering anode as the working electrode (WE) and cathodeas the counter electrodes (CE) against Ag/AgCl reference electrode(RE).

3. Results and discussions

3.1. Electricity

Fig. 2a and b show the polarization curves and power densitiesof aerobic and anaerobic SMFC reactors, respectively at day 9. Themaximum open-circuit voltage (OCV) of aerobic SMFC reactors(i.e., AR3 and AR4) and the aerobic control (i.e., AR2) were foundto be 0.72 ± 0.02 and 0.82 V, respectively, and 0.61 ± 0.04 and0.50 V for the duplicated anaerobic SMFC reactors (i.e., AnR3 andAnR4) and the anaerobic control (i.e., AnR2), respectively. In aero-bic environment, the maximum power density of the duplicatedaerobic SMFC reactors (i.e., AR3 and AR4), the aerobic control(i.e., AR2), anaerobic SMFC reactors (i.e., AnR3 and AnR4) and theanaerobic control (i.e., AnR2) were 5.77 ± 1.14, 8.67, 1.98 ± 0.5and 3.30 mW/m2, respectively. These significant differencesbetween aerobic and anaerobic SMFC performances could be dueto two main reasons. Firstly, fewer electrons were transferredthrough the external circuit since a portion of electrons releasedfrom the oxidation process were consumed by thenitrate-reducing and sulfate-reducing bacteria. Secondly, less

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Fig. 2. Electricity performance of the SMFC reactors. (a) Polarization curve (PC) and power density (PD) of the aerobic SMFCs, (b) polarization curve (PC) and power density(PD) of the anaerobic SMFCs, (c) current density (CD) of the aerobic reactors (AR2, AR3 and AR4), (d) power density (PD) of the aerobic reactors (AR2, AR3 and AR4), (e) currentdensity (CD) of the anaerobic reactors (AnR2, AnR3 and AnR4) and (f) power density (PD) of the anaerobic reactors (AnR2, AnR3 and AnR4).

M. Sherafatmand, H.Y. Ng / Bioresource Technology xxx (2015) xxx–xxx 5

energy was available with nitrate than oxygen as indicated by alower redox potential for nitrate (NO3

�/0.5N2, E0 = 0.74 V) than oxy-gen (0.5O2/H2O, E0 = 0.82 V) (Logan, 2008). Since the surface area ofanodes and cathodes were similar in this experiment, it did notmatter which one was used for the normalization. The calculatedinternal resistances (IR), based on the slope of the polarizationcurves (Fig. 2a and b), were 437 ± 70, 337, 522 ± 2 and 900 O forthe duplicated aerobic SMFC reactors (i.e., AR3 and AR4), aerobiccontrol (i.e., AR2), duplicated anaerobic SMFC reactors (i.e., AnR3and AnR4) and anaerobic control (i.e., AnR2), respectively. The rea-son behind this difference between AR2 and the duplicated aerobicSMFC reactors (i.e., AR3 and AR4) or between AnR2 and the dupli-cated anaerobic SMFC reactors (i.e., AnR3 and AnR4) could be dueto complex compounds (PAHs) that were present in the AR3, AR4,AnR3 and AnR4. In other words, with all experimental conditionsbeing similar except the absence or presence of PAHs in theSMFC reactors, the differences in electricity generation by the

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment mcarbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech

control and SMFC reactors with PAHs could probably be attributedto the different dominant microbial communities that had beendeveloped in the control and the SMFCs reactors with PAHs.With different microbial communities, the rate of electron produc-tion, due to different proton pumping rate, would be different.Therefore, higher electrical performance and lower internal resis-tance were resulted in AR2. Although there was a differencebetween the duplicated aerobic SMFC reactors (i.e., AR3 and AR4)and the aerobic control (i.e., AR2) or between the duplicated anaer-obic reactors (i.e., AnR3 and AnR4) and the anaerobic control (i.e.,AnR2), all their internal resistances were much lower than thosein the literature reported by Rezaei et al. (2007) (1297 O forChitin 20 and 1762 O for chitin), Logan et al. (2006) and Chenget al. (2006a). Therefore, although the internal resistances in thisstudy were significantly lower than others reported in the litera-ture, unlike the expectation, maximum power generation(5.8 mW/m2 for the aerobic SMFCs and 2 mW/m2 for the anaerobic

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SMFCs) was not remarkable. This could be due to the fact that nocatalyst was applied in the cathodes of the SMFC reactors used inthis study, as it has been shown by Logan and others that catalystwould increase the power generation significantly (Cheng et al.,2006b; Zhao et al., 2005).

Fig. 2c–f present the current and power densities of the SMFCreactors over 45 d. As can be seen from all figures, the duplicatedSMFC reactors (i.e., AR3, AR4, AnR3 and AnR4) had a differenttrends than those of the control rectors (i.e., AR2 and AnR2) or pre-vious SMFC performances reported by Hong et al. (2010). In eitherthe control reactors (i.e., AR2 and AnR2) or the literature, currentdensity (or power density) decreased significantly after 10, 12 or27 d, respectively, while in the duplicated SMFC reactors (AR3,AR4, AnR3 and AnR4), it remained between 16.33 and20.77 mA/m2 for the aerobic SMFC reactors and between 15.13and 17.20 mA/m2 for the anaerobic SMFC reactors over the whole45 d. However, by comparing the difference between the controlreactors (i.e., AR2 and AnR2) and the duplicated SMFC reactors(i.e., AR3, AR4, AnR3 and AnR4), the impact of PAHs on the SMFCreactors were significant – PAHs were been degraded and beenconsumed as a substrate. The minimum fluctuation in the current(or power) density observed in the AR3, AR4, AnR3 and AnR4(Fig. 2) suggested that PAHs had been removed at a constant rate.This observation was confirmed with the removal rate of PAHsmeasured using GC/MS as discussed in the next section.

3.2. PAHs removal

The analysis of PAHs biodegradation in the control SMFC reac-tors (AR1 and AnR1) and the duplicated SMFC reactors (i.e., AR3,AR4, AnR3 and AnR4) showed the effect of SMFCs on biodegrada-tion rate in both aerobic and anaerobic types of SMFC reactors.By sampling three times over 45 d from different parts of anodecompartments of the aerobic SMFC reactors (i.e., AR1 to AR4) atday 10, 25 and 45 and two times from the anaerobic SMFC reactors(i.e., AnR1 to AnR4) at day 0 and 45, and comparison with the ini-tial PAH concentration, a significant removal was of PAHs wasobserved in the sediment samples in the both aerobic and anaero-bic cathodic conditions. The results are summarized in Table 3 andFig. 3.

As it can be seen in Table 3 and Fig. 3, there was a significantdifference in the rate of PAH removal between the control SMFCreactors (i.e., AR1 and AnR1) and the duplicated SMFCs (i.e., AR3,AR4, AnR3 and AnR4). It showed the effectiveness of electrochem-ical systems for biodegradation of PAHs, as demonstrated by theAR3, AR4, AnR3 and AnR4. In aerobic condition, the duplicatedSMFC reactors (i.e., AR3 and AR4) were able to remove41.7 ± 1.7%, 34 ± 0.3% and 36.2 ± 0.9% of initial naphthalene, ace-naphthene and phenanthrene, respectively, while those of theAR1 were 12.6%, 9.8% and 11.3%, respectively. Therefore, in anaer-obic condition, the duplicated SMFC reactors (i.e., AnR3 and AnR4)were able to remove 76.9 ± 0.12%, 52.5 ± 0.04% and 36.8 ± 0.04% ofinitial naphthalene, acenaphthene and phenanthrene, respectively,

Table 3PAHs removal efficiency (%) in the SMFC and non-SMFC reactors.

PAHs compound Aerobic

Duplicated SMFCs (AR3 & AR4) Non-SMFC cont

D10 D25 D45 D10 D25

Naphthalene 13.5 ± 4.7 25.8 ± 1.6 41.7 ± 1.7 2.0 8.1Acenaphthene 12.1 ± 1.7 23.3 ± 1.1 34.1 ± 0.3 1.5 5.2Phenanthrene 8.2 ± 1.3 19.9 ± 1.8 36.2 ± 0.9 2.2 6.2

D10: Sample collected on day 10.D25: Sample collected on day 25.D45: Sample collected on day 45.

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment mcarbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech

while those of the AnR1 were 12.6%, 9.8% and 11.3%, respectively.In other words, it can be inferred that the microbial communitiesthat were formed on the surface of electrodes in the anode cham-ber could stimulate the biodegradation of PAHs. Since anaerobiccondition was maintained in the anode chamber (sediment), onlyanaerobic microorganisms were responsible for the biodegradationof PAHs.

Another interesting result from Table 3 and Fig. 3g was the sig-nificant difference between the performance of aerobic and anaer-obic SMFC reactors in the biodegradation of PAHs. For instance, theduplicated aerobic SMFC reactors (i.e., AR3 and AR4) were able toremove 41.7 ± 1.7%, 34 ± 0.3% and 36.2 ± 0.9% of the initial naph-thalene, acenaphthene and phenanthrene present, respectively,while those of the duplicated anaerobic SMFC reactors (i.e., AnR3and AnR4) were 76.9 ± 0.12%, 52.5 ± 0.04% and 36.8 ± 0.04%,respectively. This significant difference could be due to the factthat PAHs could be degraded under anaerobic conditions, in thepresence of TEAs such as nitrate and sulfate (Coates et al., 1997;Coates, 1996; Meckenstock et al., 2000; Zhang et al., 2000). ThePAH removal rates by the control SMFC reactors also confirmedthis claim since the AR1 was able to remove 12.6%, 9.8% and11.3% of the initial naphthalene, acenaphthene and phenanthrenepresent, respectively, while those of the AnR1 was 29.3%, 29.0%and 12.3%, respectively. However, it was observed that PAHs aremore susceptible to biodegradation in anaerobic SMFCs withTEAs such as nitrate and sulfate, rather than in aerobic SMFCs.This is because nitrate and sulfate not only served as the electronacceptors of PAHs degradation in the cathodic chamber, but alsoin the anodic chambers of the SMFCs. This observation has alsobeen reported in literature, whereby nitrate- andsulfate-reducing bacteria enhanced PAHs biodegradation (Coateset al., 1997; Coates, 1996; Meckenstock et al., 2000; Zhang et al.,2000). Fig. 4 shows the voltammetric cycle (CV) of the aerobicand anaerobic SMFC reactors (i.e., AR3 & AnR3) that showed goodredox activities in both the aerobic and anaerobic SMFC reactors.

Ion chromatograph (IC) analysis of the initial sediment and lakewater showed that there was negligible amount of other electronacceptors such as nitrate, sulfate and Mn (IV) present. As men-tioned earlier, nitrate and sulfate were added as potential TEAsto the anaerobic SMFC reactors. After monitoring all the ions espe-cially these two, it was found out that nitrate was the dominantTEA as it was consumed by 38.7%, while it was 13.2% for sulfate.The lower rate of reduction of sulfate compared to nitrate showedthat sulfate-reducing bacteria would take a longer time to adaptthan nitrate-reducing bacteria. It was postulated that PAHs coulddiffuse out of the sediment into the overlaying water, wherebiodegradation then occurred. However, GC analysis for PAHs inthe overlaying water in all the SMFC reactors showed that allPAHs concentrations were negligible (Fig. 3).

The difference of biodegradation rates of PAHs compounds indi-vidually in aerobic or anaerobic cathodic conditions showed thedifferent PAH biodegradation capability of microorganisms andalso, different properties of PAHs in terms of bioavailability.

Anaerobic

rol (AR1) Duplicated SMFCs (AnR3 & AnR4) Non-SMFC control (AnR1)

D45 D45 D45

12.6 76.9 ± 0.12 29.3 ± 0.059.8 52.5 ± 0.04 29.0 ± 0.03

11.3 36.8 ± 0.04 12.3 ± 0.03

icrobial fuel cells (SMFCs) for bioremediation of polycyclic aromatic hydro-.2015.06.002

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Fig. 3. PAHs biodegradation performance of the SMFC reactors. PAHs concentration of all aerobic reactors at the first and the end of the operation; (a) naphthalene, (b)acenaphthene, and (c) phenanthrene. PAHs concentration of all anaerobic reactors at the first and the end of the operation; (d) naphthalene, (e) acenaphthene, and (f)phenanthrene. (g) PAHs removal ratio (C/C�) of the duplicated reactors in both aerobic and anaerobic condition.

M. Sherafatmand, H.Y. Ng / Bioresource Technology xxx (2015) xxx–xxx 7

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment microbial fuel cells (SMFCs) for bioremediation of polycyclic aromatic hydro-carbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.06.002

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Fig. 4. Voltammogram profile of the duplicated reactors (AR3 and AnR3) at 25 mV.

8 M. Sherafatmand, H.Y. Ng / Bioresource Technology xxx (2015) xxx–xxx

Simpler compounds are typically being degraded much easier andmore rapidly, For instance, in this study, the removal efficiencies ofnaphthalene (41.7% in aerobic SMFCs and 76.9% in anaerobicSMFCs) were much higher compared to those of the acenaphthene(34.1% in aerobic SMFCs and 52.5% in anaerobic SMFCs) or phenan-threne (36.2% in aerobic SMFCs and 36.8% in anaerobic SMFCs)because naphthalene is a much simpler organic compound. Onthe other hand, this claim could not be generalized as acenaph-thene as a simpler compound compared to phenanthrene metlower removal rate in aerobic SMFC reactors.

3.3. Total organic carbon (TOC)

Total organic carbons (TOC) were monitored in the water andsediment samples during the process to determine the biodegrada-tion of PAHs by the SMFC reactors. Fig. 5 shows the differences ofTOC/TOC� ratio in the SMFC reactors during the operation in theaerobic condition. It was found that this ratio for the duplicatedaerobic SMFC reactors (i.e., AR3 and AR4) was higher than that ofthe AR1 (control 1, which was a non-SMFC reactor with PAHs con-taminated sediment) and AR2 (control 2, which was a SMFC withnon-polluted sediment). The same trend was observed for theanaerobic SMFC reactors. Comparison of the AR1 and AnR1 withthe duplicated SMFC reactors (i.e., AR3, AR4, AnR3 and AnR4)showed that on Day 45%, 52% and 67% of the initial TOC were con-sumed by the microorganisms in the duplicated aerobic SMFCs(AR3 and AR4) and anaerobic SMFCs (AnR3 and AnR4), respec-tively, while only 37% and 31% of the initial TOC were removedin the AR1 and AnR1, respectively. It means that microorganismshad been more active in the SMFC reactors and consequently, more

Fig. 5. TOC/TOC� ratio of the aerobic reactors (sediment) during the operation. D10:sample collected on day 10. D25: sample collected on day 25. D45: sample collectedon day 45.

Please cite this article in press as: Sherafatmand, M., Ng, H.Y. Using sediment mcarbons (PAHs). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech

PAHs had been degraded in SMFC reactors compared to thenon-SMFC reactor. When the results of AR2 and AnR2 were com-pared to those of the duplicated reactors (i.e. AR3, AR4, AnR3 andAnR4), the presence of PAHs in the sediment matrix served asthe organic substrate for the SMFC reactors, achieving TOC removalof 52% and 67% in the aerobic and anaerobic reactors on Day 45,while only 27% and 41% of the initial TOC were consumed in AR2and AnR2. Better efficiency in TOC removal obtained from anaero-bic reactors could be due to that nitrate and sulfate could be uti-lized as TEAs in the soil for more degradation of carbon sources.

To date, bioaugmentation and biostimulation are widely knowntechnologies to remediate hydrocarbon-polluted sites(Amezcua-Allieri et al., 2012), whereby the addition of nutrientsor microorganisms is required. Recently, in situ remediation ofcontaminated soils has received considerable attention due to itsmany advantages such as low cost and the avoidance of secondarypollution (Huang et al., 2011; Rulkens, 2005). However, this studyhad demonstrated a new way of increasing the rate of PAHs reme-diation by harvesting the electrons generated via an external cir-cuit and stimulation of microorganism by means of a SMFC inboth aerobic and anaerobic cathodic environments. This stimula-tion could be a result of altering the physical and chemical of soilproperties by applying a potential difference and more activatedmedium for microorganisms provided by the SMFCs. This assertionthat a SMFC has the ability of changing the physio-chemical prop-erties of sediment organic matter (SOM) has been already investi-gated by Hong et al. (2010). In other words, SMFCs may stimulateindigenous microorganisms to make them more active for electrongeneration and transferring. This study suggested that SMFC couldbe a potential bioremediation technology for in situ remediation ofsoils or sediments contaminated with PAHs.

4. Conclusion

SMFC was found capable of biodegrading complex PAHs in sed-iment. The SMFCs achieved 41.7%, 31.4% and 36.2% PAHs removalin aerobic environment and 76.9%, 52.5% and 36.8% in anaerobicenvironment for naphthalene, acenaphthene and phenanthrene,respectively. In addition, this study also showed that SMFCs canstimulate TOC removal in sediment. The SMFCs showed 52% and67% TOC removal from the sediment, while it was only 27% and41% for the non-SMFC reactor in the aerobic and anaerobic reac-tors, respectively. This ability demonstrated the potential of BESsin removing higher molecular weight and recalcitrant PAHs, creat-ing new prospects of in situ bioremediation.

Acknowledgement

This research was supported by National University ofSingapore Grant, SINGA program.

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