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Bioremediation Journal

ISSN: 1088-9868 (Print) 1547-6529 (Online) Journal homepage: http://www.tandfonline.com/loi/bbrm20

Solar power enhancement of electrokinetic bioremediation of phenanthrene by Mycobacterium pallens Ikrema Hassan, Eltayeb Mohamedelhassan, Ernest K. Yanful & Ze-Chun Yuan To cite this article: Ikrema Hassan, Eltayeb Mohamedelhassan, Ernest K. Yanful & Ze-Chun Yuan (2017) Solar power enhancement of electrokinetic bioremediation of phenanthrene by Mycobacterium pallens, Bioremediation Journal, 21:2, 53-70, DOI: 10.1080/10889868.2017.1312264 To link to this article: http://dx.doi.org/10.1080/10889868.2017.1312264

Published online: 04 May 2017.

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Date: 09 May 2017, At: 11:21

BIOREMEDIATION JOURNAL 2017, VOL. 21, NO. 2, 53–70 http://dx.doi.org/10.1080/10889868.2017.1312264

Solar power enhancement of electrokinetic bioremediation of phenanthrene by Mycobacterium pallens Ikrema Hassana,b, Eltayeb Mohamedelhassanc, Ernest K. Yanfula, and Ze-Chun Yuanb,d a Department of Civil and Environmental Engineering, University of Western Ontario, London, Ontario, Canada; bLondon Research and Development Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada; cDepartment of Civil Engineering, Lakehead University, Thunder Bay, Ontario, Canada; dDepartment of Microbiology and Immunology, Schulich School of Medicine, University of Western Ontario, London, Ontario, Canada

ABSTRACT

Enhanced bioremediation of phenanthrene-contaminated soil with Mycobacterium pallens was conducted. Kaolinite was used in the tests as a soil matrix and was artificially contaminated with phenanthrene at a concentration of 2 mg phenanthrene per gram dry soil. Mycobacterim pallens at concentration of 108 colony-forming units (CFU) per milliliter was used as a potential microorganism to degrade phenanthrene. Aspects of the study included evaluating efficacy of using Mycobacterium pallens for degrading phenanthrene, electrokinetics for delivering nutrients and microorganisms to contaminated soil, and solar panels for generating power for electrokinetic bioremediation. A novel anode-cathode configuration, in which the anode and cathode are placed in the same compartment, was implemented to control/minimize changes in pH during electrokinetic bioremediation. The nutrients (NO3¡), electrical current, temperature, Mycobacterium pallens (CFU), and phenatherene concentration were evaluated. The results showed that solar panels generated sufficient power for electrokinetic bioremediation. The highest current obtained was generated when bacteria and nutrients were added to the soil. This was associated with the highest phenanthrene removal from the soil (50% of the initial concentration). Additionally, we determined that the novel anode-cathode configuration in the electrokinetic bioremediation cell was successful in delivering the bacteria and nutrients to the contaminated soil and in maintaining a relatively neutral pH around the electrode compartments, which improved the remediation. Overall, this study showed that the use of solar power with electrokinetic bioremediation can provide a cost-effective approach to reduce and remove hydrocarbon contaminations in soil.

KEYWORDS

Bioremediation; electrokinetics; contaminated soil; pH stabilization; phenanthrene; solar energy

Introduction Polycyclic aromatic hydrocarbon (PAH) compounds are usually found in crude oil, coal, and tar deposits, and they are released into the environment via accidental spills, leakage from underground storage tanks and pipelines, and industrial as well as agricultural activities. PAH compounds are known to be toxic, mutagenic, teratogenic, and/or carcinogenic (Reddy and Saichek 2003; Reddy and Cameselle 2009). Therefore, when released to the environment, PAHs become a health hazard to humans and a risk to ecological receptors. In this study, phenanthrene was selected as a model compound to represent hydrocarbon contaminants because it is one of the 16 PAH compounds

listed by the United States Environmental Protection Agency (EPA) as priority pollutants (Bouvrette et al. 2006; Andersson and Achten 2015). Phenanthrene, which is composed of three fused benzene rings, is known as a parent compound because it is dominant in the chemical structure of many PAHs. Many researchers have used phenanthrene because it is the simplest compound that contains both a bay region and k region (Cerniglia 1984; Boldrin, Tiehm, and Fritzsche 1993; Zhao et al. 2009), which are used to determine the relative carcinogenetic properties of PAH compounds (Puglisi et al. 2007). Many recalcitrant heavy hydrocarbons contain four benzene rings

CONTACT Ze-Chun Yuan [email protected], [email protected] London Research and Development Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, N5V 4T3 Canada; Ernest K. Yanful [email protected] Department of Civil and Environmental Engineering, University of Western Ontario, London, Ontario, N6A 5B9, Canada. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bbrm. © 2017 Taylor & Francis

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or more that can be degraded via co-metabolic degradation. For example, some microorganisms are able to utilize phenanthrene (three benzene rings) as their sole carbon and energy source and can produce enzymes that can degrade other hydrocarbon compounds, such as fluorene (Boldrin, Tiehm, and Fritzsche 1993; Puglisi et al. 2007; Coppotelli et al. 2010). Many remediation techniques have been developed and implemented to mitigate contaminated sites. These techniques include pump and treat, thermal desorption, aeration, biopiles, electrokinetics, soil vapor extraction, soil washing, and soil flushing, and among these, bioremediation technology stands out for its low cost and minimal negative impact on the environment (Moody et al. 2001). Bioremediation, including phytoremediation with plants and microbemediated bioremediation, is one of the most effective remediation techniques and eco-friendly. Recent studies have investigated an innovative, hybrid technique that joins electrokinetics and bioremediation (Yeung and Gu 2011; Gill et al. 2014; Hassan et al. 2016b). Electrokinetic remediation can be defined as the implementation or application of a low-level direct current (DC) through the contaminated soil. The electric field is applied using pairs of positively charged electrodes (anodes) and negatively charged electrodes (cathodes) inserted vertically or horizontally at a certain distance between the boundaries of the soil under treatment (Acar and Alshawabkeh 1993). The applied electric field in an electrokinetic treatment process is essential to provoke three contaminant-transport mechanisms: electroosmosis, electromigration, and electrophoreses plus electrolysis reactions. Electroosmotic flow is defined as the movement of fluid in soil pores, such as water and chemicals, from a positively charged electrode (anode) towards a negatively charged electrode (cathode) that results from an applied electric field gradient. The application of an electric field in electrokinetic remediation processes exploits ion transport through the phenomenon of electromigration. Electromigration, also known as ionic migration, is the movement of ions towards oppositely charged electrodes (Koryta 1982). Electrophoresis, which is the movement of charged colloids towards an oppositely charged electrode, is only important in soil slurries and has negligible impact in compacted soil. In an electrokinetic process, the electrolysis of water occurs at the electrodes and results in oxidation-

reduction reactions (Acar and Alshawabkeh 1993). Oxidation takes place at the anode, which generates hydrogen ions (HC) and liberates oxygen gas, as in Equation 1: 2H2 O C 4e ¡ ! O2 " C 4H C

(1)

Reduction occurs at the cathode, which produces hydroxyl ions (OH¡) and disperses hydrogen gas, as in Equation 2: 2H2 O C 2e ¡ ! H2 " C 2OH ¡

(2)

The generated hydrogen ions contribute to an overall decrease in the pH of the environment near the anode, whereas the hydroxyl ions elevate the pH at the vicinity of the cathode. Typically, the pH reaches as low as 2 at the anode and as high as 11 at the cathode (Page and Page 2002). Bacteria can survive in a wide range of pH environments; however, the biodegradation efficiency is highest within a narrow pH range, and neither low nor high pH levels favor the bioremediation process. Many studies have investigated and implemented various techniques to control or minimize the pH changes during electrokinetic remediation (Wong, Hicks, and Probstein 1997; Kim and Han 2003; Chen et al. 2006; Niqui-Arroyo et al. 2006; Mao et al. 2012; Rajic et al. 2012; Wu et al. 2012). In this study, in order to stabilize pH during electrokinetic bioremediation, we implemented a new technique in which the electrokinetic cell was divided into three compartments, with a soil compartment in the middle and one water compartment on each side. Each water compartment housed an anode and a cathode, and it was predicted that the coexistence of an anode and a cathode in the same water compartment would cause the hydrogen ions generated at the anode and the hydroxyl ions produced at the cathode to neutralize each other by forming water and therefore keeping the pH relatively balanced. In addition, the hybrid approach of electrokinetic bioremediation can potentially employ the transport mechanisms associated with electrokinetics to accelerate the natural biodegradation of contaminants; such enhanced transport would increase the opportunities for interactions between microorganisms and contaminants while stimulating the existing microbial community in the subsurface by delivering nutrients that promote

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microbial growth (Acar, Rabbi, and Ozsu 1997; Budhu et al. 1997). Many bacterial strains from the genus Mycobacterium are capable of degrading phenanthrene, including Mycobacterium vanbaalenii PYR-1 and Mycobacterium sp. LB501T and CABI (Boldrin, Tiehm, and Fritzsche 1993; Moody et al. 2001; Kim et al. 2005; Seo, Keum, and Li 2009; Zeng et al. 2010). Two Mycobacterium species, NJS-1 and NJS-P, were found capable of degrading 90% and 50% of pyrene and fluoranthene, respectively, over 2 months (Kim et al. 2008). Mycobacterium pallens can also degrade pyrene and fluoranthene (Hennessee et al. 2009) and was first isolated from noncontaminated soil in Wahiawa, Hawaii, USA. M. pallens is a nonmotile aerobe, forms colonies in 7 days, and grows well at 28 C and 37 C. The success of the genus Mycobacterium in degrading PAH can be attributed to their diverse array of degradative enzymes. For instance, M. vanbaalenii PYR-1 possesses the dioxygenases NidAB and NidA3B3 (Nikle 3,3' Diaminobenzidine), which can convert a wide range of PAH into cis-dihydrodiols (Kim et al. 2008). The operating systems of the available remediation techniques currently used in laboratory or field remediation require energy to execute their processes, with the energy consumption varying from one technique to another. In general, high-energy consumption increases the overall cost of the remediation processes and can become a major obstacle that restricts field applications of the technologies. In the last decade, the need for finding economical alternative sources of power to replace nonrenewable sources has become urgent. Solar energy, a renewable alternative to fossil fuels with no adverse environmental impact, has gained the attention of scientists and the general public, leading to a multitude of beneficial applications (Yuan et al. 2009; Hassan, Mohamedelhassan, and Yanful 2015; Hassan et al. 2016b). The use of solar panels to generate power can reduce electricity transmission expenses and eliminate power loss in transmission, in particular, for remote and isolated areas where the electricity grid is not available. Solar panels produce a direct current (DC) electric field that is acceptable in electrokinetic applications without alteration, i.e., without the need for a DC transformer to the alternating current (AC). Therefore, electrokinetic bioremediation is an excellent candidate for utilizing solar panels because there is no need for the conversion process from DC to AC. Although solar

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panels can be an excellent power supply for remediation techniques, there is little to no research in the available literature on the use of solar panels in electrokinetic bioremediation or on the effect of the off power cycle (during darkness) on microorganisms. The present study investigated the use of electrokinetic bioremediation to mitigate the decontamination of kaolinite clay artificially contaminated with phenanthrene. The main objectives of this study were to (i) investigate the use of solar panels to generate power for electrokinetic bioremediation; (ii) assess the ability of M. pallens to degrade phenanthrene; (iii) evaluate the effectiveness of electrokinetics for stabilizing pH during electrokinetic bioremediation of phenanthrene-contaminated soil; and (iv) examine the efficiency of our novel electrode configuration in delivering nutrients and bacteria compared with the conventional anode-cathode configuration.

Materials and methods Soil matrix (clay)

Inorganic kaolinite clay, 96% to 99.9% kaolinite (Edgar Minerals, Hawthorne, FL, USA), was used as the soil matrix for the electrokinetic bioremediation experiments. The clay soil has a very small pore size, a long, tortuous flow path, and subsequently low hydraulic permeability. Thus, it is extremely difficult to perform in situ remediation for contaminated clay soil using traditional remediation methods such as soil flushing. The transport of pore fluids by electrokinetics is largely independent of pore size; therefore, electrokinetic remediation has the capacity to remediate contaminated clay soils where other remediation technologies are rendered ineffective. Table 1 lists the physical and chemical properties of the soil. Atterberg limits (liquid and plastic limits) were determined following procedure D4318-10 (ASTM 2010) described by the American Society for Testing and Materials (ASTM). The maximum and the minimum void ratios were determined. The maximum void ratio (emax) is the void ratio (i.e., volume of voids divided by the volume of solids) of the soil in its loosest state, and it was determined by allowing a soil sample to settle by gravity in a graduated cylinder, covering the cylinder with a latex sheet, turning the cylinder upside down slowly, and then

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Table 1. Soil physiochemical properties. Soil property Liquid limit Plastic limit emax emin pH Cation-exchange capacity Specific surface area

Measured value 60 (§ 2) 34 (§ 0.5) 3.1 (§ 0.4) 0.87 (§ 0.02) 4.2 (§ 0.1) 3.75 (§ 0.10) mEq/100 g of soil 28.75 (§ 1.5) m2/g

Note. The values in parentheses indicate the standard deviation. Tests were conducted in triplicates.

measuring the soil volume (Venkatramaiah 1995; Yamamuro and Lade 1997; Wood 2004). The minimum void ratio (emin) is the void ratio of the soil in its densest state and was determined by measuring the volumes of soil voids and solids after compaction using the modified Proctor test (Venkatramaiah 1995; Wood 2004; Bradshaw and Baxter 2007). Five water contents, 0.14, 0.18, 0.30, 0.35, and 0.48, were used in the compaction test. Sieve analysis on the soil revealed that all the particle sizes were less than 0.075 mm (passed No. 200 sieve). Accordingly, hydrometer analysis was conducted to obtain particle size distribution, in accordance with ASTM D422-63 (ASTM 2007). A total organic carbon analyzer (TOC-VCPN; Shimadzu, Kyoto, Japan) was used to determine the organic carbon fraction (foc) in the soil. Soil pH was determined using ASTM D4972-13 (ASTM 2013). To determine the cation-exchange capacity, ammonium acetate and potassium chloride were used as extractants to obtain the soluble cations and then bound or exchangeable cations. Inductively coupled plasma optical emission spectroscopy (ICP-OES) (Varian, Palo Alto, CA, USA) was used to determine the cation concentrations in solution. The specific surface area of the kaolinite clay was determined using a Gemini surface area analyzer (Micromeritics, Norcross, GA, USA). The specific area was estimated using the BrunauerEmmett-Teller (BET) method, in which the surface area is calculated from the amount of nitrogen gas adsorbed by the soil particles measured at the boiling point of nitrogen and atmospheric pressure. Phenanthrene, chemical compounds, and solvents

Phenanthrene, 99.7% purity, was purchased from Fisher Scientific (Ottawa, ON, Canada). All solvents

used (methanol, acetone, dichloromethane) were high-performance liquid chromatography (HPLC) grade and purchased from Sigma Aldrich (Oakville, ON, Canada). Chemical compounds NaCl, NaNO3, and KH2PO4 were also purchased from Sigma Aldrich. Bioremediation bacterium

Mycobacterim pallens ATCC 1372 (Hennessee et al. 2009) was obtained as a freeze-dried pellet from Cedarlane (Burlington, ON, Canada; original source: American Type Culture Collection [ATCC], Manassas, VA, USA), and the frozen pellet was rehydrated and cultured as recommended by ATCC. Bacterial pellets for stock were washed two times using normal saline (0.85% NaCl) (w/v), resuspended in 0.85% (w/ v) NaCl and 15% (v/v) glycerol, snap-frozen using liquid nitrogen, and then stored at ¡80 C. For routine culturing, 1395 liquid medium and 7H10 agar medium (VWR, Mississauga, ON, Canada) were used. The 1395 medium was prepared using 4.7 g Middlebrook 7H9 broth (VWR) (MiddleBrook 7H9 Broth ingredients per 900 ml of deionized water: disodium phosphate 2.5 g, monopotassium phosphate 1.0 g, Lglutamic acid 0.5 g, sodium citrate 0.1 g, magnesium sulfate 50.0 mg, ferric ammonium citrate 40.0 mg, zinc sulfate 1.0 mg, copper sulfate 1.0 mg, pyridoxine 1.0 mg, calcium chloride 0.5 mg, biotin 0.5 mg), 2.0 ml glycerol, and 900 ml deionized water, supplemented after autoclaving with 100 ml of sterile albumin-dextrose-catalase (ADC) growth supplement (ADC enrichment ingredients per 100 ml deionized water: bovine albumin 5.0 g, dextrose 2.0 g, beef catalase 3.0 mg). Minimal medium contains the following: NH4SO4 1 g/L, KH2PO4 0.5 g/L, K2HPO4 0.5 g/L, MgSO4 0.1 g/L, NaCl 6 g/L, CaCL2 0.1 g/L, MES hydrate 1 g/L, 15 g/L agar, pH 5.75.

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Electrokinetic apparatus

The experimental equipment consisted of three custom-made, electrokinetic remediation cells (Sabic Polymershapes, London, ON, Canada), a custom-made, electrokinetic voltage controller (EKVC) (Electronic Shop, University of Western Ontario), solar panels (Sanyo Corporation, Osaka, Japan), a data acquisition system (USB-6008; National Instruments, Austin, TX, USA), portable data logging system (Omega 320; London, UK), Eppendorf centrifuge 5810R 15-amp version (Hamburg, Germany), Bio-Rad SmartSpec Plus (Hercules, CA, USA), and gas chromatography– mass spectrometer (GC-MS) (Agilent Technologies, Santa Clara, CA, USA). A solar panel was used to generate the power for the electrokinetic bioremediation. The solar panel dimensions were 1590 mm £ 820 mm. The maximum current and maximum power of the solar panel were 3.40 A and 200 W, respectively. The electrokinetic bioremediation (EKB) cells, constructed from clear Plexiglas plates 12 mm in thickness, had inner dimensions of 350 £ 120 £ 100 mm (length £ width £ height), as shown in Figure 1. Each EKB cell was composed of an upper part, base, cover, and two movable, rectangular, perforated Plexiglas plates (120 mm £ 100 mm). The EKB cell was divided by the rectangular perforated Plexiglas into two water compartments with a soil specimen chamber in between. Each water compartment housed an anode and a cathode (Figure 1). The first water compartment hosted Anode A1C and Cathode A2¡, and the second compartment contained Anode A2C and Cathode A1¡. In each EKB cell, the electrodes were connected to the solar panel using two electric circuits (Figure 1): circuit 1 between Anode A1C and Cathode A1¡, and circuit 2 between Anode A2C and Cathode A2¡ (in the second and third EKB cells, electric circuit 1 was between B1C and B1¡ and C1C and C1¡ and electric circuit 2 connected B2C to B2¡ and C2C to C2¡). It was predicted that the coexistence of an anode and a cathode in the same water compartment would cause a neutralization reaction between hydrogen ions generated at the anode and the hydroxyl ions produced at the cathode, thereby forming water and keeping the pH unchanged (Hassan et al. 2016a). The electrokinetics voltage controller (EKVC) device (Figure 1) was designed to switch the electric potential between the two electric circuits at predetermined intervals, such that at any given time, there is

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only one electric current running through either electric circuit 1 or 2 in the EKB cell. The EKVC takes a maximum current of 1 A and an input DC voltage of up to 70 V, with six output ports and a programmable timer. A computer code was written using LabVIEW 2013 (National Instruments) to connect the data acquisition terminals and EKVC with a personal computer (PC) for real-time data recording. The EKVC alternates voltage between outputs groups at a set programmable time. Procedures for phenanthrene degradation

Preliminary tests were conducted to investigate the ability of M. pallens to degrade phenanthrene. M. pallens was grown in agar plates coated with phenanthrene prepared as follows. Agar plates, containing approximately 10 ml of Middlebrook 7H10 medium (MiddleBrook 7H10 agar ingredients per 995 ml of deionized water: disodium phosphate 1.5 g, monopotassium phosphate 1.5 g, L-glutamic acid 0.5 g, ammonium sulfate 0.5 g, sodium citrate 0.4 g, ferric ammonium citrate 40.0 mg, magnesium sulfate 25.0 mg, zinc sulfate 1.0 mg, copper sulfate 1.0 mg, pyridoxine 1.0 mg, calcium chloride 0.5 mg, biotin 0.5 mg, Malachite Green 0.25 mg, glycerol 5.0 ml, agar 15.0 g) supplemented with Dubos oleic-albumin complex (OADC) (OADC enrichment ingredients per 100 ml deionized water: bovine albumin 5.0 g, dextrose 2.0 gm, sodium chloride 0.85 g, beef catalase 4.0 mg, oleic acid 50.0 mg), were spread with 100 ml of a stock solution of 100 mg/ml phenanthrene prepared in dimethyl sulfoxide (DMSO); the phenanthrene formed an opaque layer, and plates were left under a biosafety cabinet to allow for DMSO evaporation. M. pallens was grown in 1395 medium at 30 C for 7 days, pelleted by centrifugation at 10,000 rpm for 10 min, washed with 5 ml sterile 0.85% (w/v) NaCl twice to remove nutrients, and resuspended and diluted in 0.85% (w/v) NaCl to an optical density (OD600) of 1. Inocula (20 ml) from this solution were added to the center of the phenanthrene-coated agar plates, which were then incubated at 30 C and monitored for a clearing zone around the center. The test was conducted in triplicate. Procedures for electrokinetic bioremediation

The physical and chemical properties of the inorganic kaolinite clay soil are presented in Table 1. Soil samples with a phenanthrene concentration of 2 mg/g soil

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Figure 1. Schematic diagram of the electrokinetic bioremediation (EKB) cell, EKVC, Omega 320, and solar panel.

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were prepared as follows. The soil was sterilized for 24 h at 120 C in a dry oven, and then to verify sterilization, 1 g of soil was mixed with 10 ml of 0.85% NaCl (sterile), left to settle by gravity, and serial dilutions of the supernatant were prepared, plated on Luria-Bertani (LB) agar (LB ingredients: 10.0 g/L tryptone, 5.0 g/L yeast extract, 5.0 g/L sodium chloride, 1 L double-distilled [dd] H2O, 15 g/L agar) plates and visually inspected for bacterial growth. The sterile soil and the corresponding amount of phenanthrene (phenanthrene was dissolved in methanol) were thoroughly mixed using a mechanically rotating rod. To evaporate the methanol, the mixture was placed under a fume hood overnight, in the dark to avoid degradation of phenanthrene by photo-oxidation. Stock solutions of 1g/L of NaCl, NaNO3, and KH2PO4 were prepared. Four sets of tests were conducted. Table 2 shows the details of the test setup. The first set was designed to compare the efficiency of the new anode-cathode compartment (ACC) technique and the conventional anode-cathode (CAC) technique in delivering bacteria into the clay soil. Sterilized kaolinite was saturated with sterilized, deionized water, giving a final water content of 60%, and placed in the EKB cell soil chambers (Cell X and Cell Y) in three layers to a total height of 60 mm. Each layer was tamped using a rectangular Plexiglas plate and a pestle to prevent the entrapment of air pockets and to produce soil specimens with similar densities. M. pallens (108 colony-forming units [CFU]/ml; prepared from the glycerol stock) was suspended in a solution of 0.1% (w/v) NaNO3 and 0.1% (w/v) KH2PO4 and loaded into the water compartments. The new ACC electrode configuration was used in Cell X. For Cell Y, the CAC configuration was used. In the second set (Cell A with ACC configuration), the soil was artificially contaminated with 2 mg phenanthrene per gram of dry soil. A soil sample with

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a water content of 60% was prepared using M. pallens (108 CFU/ml) suspended in 0.1% (w/v) NaCl solution and placed in the soil chamber. The water compartments were filled with a solution of 0.1% (w/v) NaNO3 and 0.1% (w/v) KH2PO4. The control test for this configuration was Cell H in which no electric field was applied. The third set was designed to represent a bioaugmentation scenario, i.e., the introduction of bacterial strain/strains with superior capabilities. In the third set of tests (Cell B with ACC configuration), the soil was artificially contaminated with phenanthrene at 2 mg/g and thoroughly mixed with a solution of 0.1% (w/v) of NaNO3 and 0.1% (w/v) KH2PO4 to a water content of 60%. The soil sample was placed in the soil chamber in layers (as described above). M. pallens (108 CFU/ml) was resuspended in 0.85% NaCl (w/v) and added to the water compartments. Cell F represented the control for the third set of tests and had no electric field applied. The fourth set was designed to simulate a contaminated site where the indigenous bacteria lacked the ability to degrade the contaminant, and the nutrients were not sufficient for bacterial growth. In the fourth set (Cell C with ACC configuration), the soil was artificially contaminated with phenanthrene (2 mg/g) and placed in the soil chamber. M. pallens (108 CFU/ml), suspended in NaNO3 and KH2PO4 solution, was added to the water compartments. The control for this set of tests was Cell G. All the experiments and tests were conducted at London Research and Development Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada (the study site). The EKB cells, a PC, and the data acquisition system were placed inside two cabinets in a microplot field area (open yard) at the London Research and Development Center (LRDC). The

Table 2. Testing parameters. Test no. 1 2 3 4 5 6 7 8

EKB cell Cell X Cell Y Cell A Cell B Cell C Cell D Control Cell E Control Cell F Control

Soil compartment No phenanthrene No phenanthrene 2 mg/g phenanthrene, M. pallens, and nutrient solution 2 mg/g phenanthrene and 1 g/L of NaNO3 and KH2PO4 2 mg/g phenanthrene 2 g/L phenanthrene, M. pallens, and nutrient solution 2 g/L phenanthrene and 1 g/L of NaNO3 and KH2PO4 2 g/L phenanthrene

Water compartment 

Mycobacterium pallens M. pallens 1 g/L of NaNO3 and KH2PO4 M. pallens in 0.85% NaCl M. pallens and 1 g/L of NaNO3 and KH2PO4 1 g/L of NaNO3 and KH2PO4 M. pallens in 0.85% NaCl M. pallens and 1 g/L of NaNO3 and KH2PO4

Note. Mycobacterium pallens (M. pallens) was used at a concentration of 108 CFU/ml. Tests were conducted in triplicates; errors: §0.25»0.5%.

Solar power EK (ACC) EK (CAC) EK (ACC) EK (ACC) EK (ACC) No No No

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latitude and the longitude of the study site are 42.98 and ¡81.24, respectively. The solar panel was tilted to an angle of 28 and faced south. Procedures for phenanthrene extraction and determination

Phenanthrene was extracted from soil samples before and after the tests, and the concentration was determined using GC-MS. Phenanthrene was extracted from soil using a sonicating water bath for 4 h at room temperature. Soil (1.0 g) was weighed in 35-ml heavy-duty glass tubes, and 20 ml of a 1:1 acetone/ dichloromethane solution was added to the glass tube and placed in the sonicating water bath for 2 h. The solution was replaced with a fresh solution and sonicated for another 2 h. After sonication, the solution was replaced for the second time with a fresh 20-ml solution, and the glass tube was placed in a shaker table (150 rpm) overnight. The solutions were then combined (60 ml), and a chromatography column packed with alumina and anhydrous sodium sulfate was used to filter the analyte. A Kuderna-Danish apparatus was used to reduce the volume (analyte 60 ml) to 5 ml. The extraction method was validated by the addition of 0.5, 1, 2, 3, 4, and 5 mg of phenanthrene to 1 g of sterile, dry soil and the phenanthrene was then extracted. The results showed that the data fit the regression line with R2 D 0.98. The extraction tests showed recovery of approximately 97% phenanthrene from an initial concentration of 2 mg/g dry soil. The final solution (5 ml analyte) was filtered using 25-mm Twin Peak filter (TPF). For determining phenanthrene concentrations, the GC-MS method used a gas chromatography column HP-MS (length 30 m, internal diameter 250 mm, film thickness 0.25 mm; Agilent Technologies, Santa Clara, CA, USA), with helium as a carrier gas, injected volume of 1 ml, and initial temperature of 150 C held for 2 min, and then ramped at 10 C per minute up to 280 C and held for 2 min. The voltage output of the solar panel, voltage gradient across the EKB cells, temperature inside the EKB cells, and the electric current through the EKB cells were monitored and recorded regularly during the test using real-time data acquisition systems described above (USB-6008; National Instruments). After the tests, the soil samples in the EKB cells were divided into four sections, S1 to S4,

Figure 2. (A) Soil sections in the conventional anode-cathode configuration (CAC). (B) Soil sections in the anode-cathode configuration (ACC).

between the anode and the cathode, and phenanthrene concentration was determined for each section (Figure 2A and B).

Results and discussion Electrical potential generated by solar panel

The tests in this study were conducted during the summer (July to August 2015). For optimum electrical potential generation, the solar panel was tilted to an angle of 28 facing south. The electrical potential generated by the solar panel and the electric current through the EKB cells were recorded in real time by the data acquisition system. The electrical potential generated by solar panels depends on the duration of daylight and varying weather conditions, which can cause fluctuations in the power supply during the day and intervals of zero voltage at night, especially in the northern hemisphere where low daylight occurs in the winter months. During the test period of 30 days, the average voltage generated by the solar

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Figure 3. Voltage generated by solar panel.

panels increased from 0 V before sunrise to a maximum voltage of 57 V around 9:00 a.m. (Figure 3). Between 9:00 a.m. and 4:00 p.m., the generated voltage ranged between 52 and 57 V and then started to decrease rapidly around 7:00 p.m., reaching zero at sunset. Figure 3 reveals a fluctuation in power generated from the solar panel during the day and a diminishment of the electric field during the night. Analogous to the positive effect of intermittent current in electrokinetic application, as reported by previous studies, the fluctuation of the power generated from the solar panel can stimulate the remediation process (Mohamedelhassan and Shang 2001; Reddy and Saichek 2004; Hansen and Rojo 2007; Reddy and Cameselle 2009). Normally, the application of an electric field in electrokinetics causes ions to orient in a double layer against the electric current, reducing the efficiency of the remediation process, but the fluctuation of the electric field generated by solar panels allows for the restoration of the original ion orientation, which can enhance the remediation process (Mohamedelhassan and Shang 2001; Hansen and Rojo 2007; Ryu et al. 2009, 2010; Jo et al. 2012; Kim et al. 2013). The voltage profile shows that the solar panel is capable of generating voltage in the range of