Microbial Fuel Cells Generating Electricity from Rhizodeposits of

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Environ. Sci. Technol. 2008, 42, 3053–3058

Microbial Fuel Cells Generating Electricity from Rhizodeposits of Rice Plants LIESJE DE SCHAMPHELAIRE,† LEEN VAN DEN BOSSCHE,† HAI SON DANG,† MONICA HÖFTE,‡ NICO BOON,† KORNEEL RABAEY,§ AND W I L L Y V E R S T R A E T E * ,† Laboratory of Microbial Ecology and Technology (LabMET) and Laboratory of Phytopathology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium, and The Advanced Water Management Centre, University of Queensland, St Lucia QLD 4072, Australia

Received August 03, 2007. Revised manuscript received January 14, 2008. Accepted January 22, 2008.

Living plants transport substantial amounts of organic material into the soil. This process, called rhizodeposition, provides the substrate for the rhizospheric microbial community. In this study, a laboratory-scale sediment microbial fuel cell, of which the anode is positioned in the rhizosphere of the rice plants, is used to microbially oxidize the plant-derived organics. An electrical current was generated through the in situ oxidation of rhizodeposits from living rice plants. The electrical power output of a sediment microbial fuel cell was found to be a factor 7 higher in the presence of actively growing plants. This process offers the potential of light-driven power generation from living plants in a nondestructive way. Sustainable power productions up to 330 W ha-1 could be attributed to the oxidation of the plant-derived compounds.

Introduction Plants continuously provide an input of organic matter to the soil throughout their plant life. As plants decay, dead roots and shoot residues remain in the soil. During the growing season, organic carbon enters the soil as rhizodeposits (1). The latter comprise several groups of organics such as (i) water-soluble, low molecular weight, and passively lost exudates, (ii) secretions which are of high molecular weight and actively lost, (iii) lysates from sloughed-off cells and decaying roots, (iv) gases, and (v) mucilage covering roots (2). Moreover, rhizodeposition also comprises the C flux that is directed out of roots through symbiotic mycorrhizal fungi, which are associated with over 80% of the plants (3). Since it is difficult to experimentally distinguish exudates from other rhizodeposits, root exudates are often defined as all organic substances released by healthy and intact roots into the environment. They comprise a gathering of carbohydrates, amino acids, amides, aliphatic acids, aromatic acids, fatty acids, sterols, enzymes, hormones, vitamins, and others (2). The release of exudates plays an important role in nutrient acquisition (4). * Corresponding author phone: 0032 9 264 59 76; fax: 0032 9 264 62 48; e-mail: [email protected]. † LabMET, Ghent University. ‡ Laboratory of Phytopathology, Ghent University. § University of Queensland. 10.1021/es071938w CCC: $40.75

Published on Web 03/18/2008

 2008 American Chemical Society

In rice paddies, rhizodeposition counts for 200 kg organic C ha-1 crop cycle-1 (5). In a flooded rice system, this substantial input of organic material is transformed into methane to the extent that rice agriculture worldwide contributes 7-20% of the total methane emissions (6). Rhizodeposition was shown to be the main origin of methane evolution in rice paddies, with a share of 25% from exudates and 75% from decomposing root residues (5). Next to being a source of greenhouse gases, these rhizospheric processes also represent a significant loss of energy from the rice system: rice plants lose substantial amounts of trapped solar energy as rhizodeposition, while the gaseous end product of the anaerobic composition thereof, methane, has a high energetic value. It would most certainly be interesting to recuperate this flow of energy from living plants, as it represents a true source of green energy. In this research, the latter was attempted through the installation of a so-called sediment microbial fuel cell (SMFC) in the rhizosphere of the rice plants. A SMFC is a microbial fuel cell, with an anode buried in a reduced matrix and a cathode floating in the overlying, oxidized water layer (7). The submerged matrix can serve as a support for plant growth; it can be composed of various types of materials. At the anode, a microbially catalyzed oxidation of reduced compounds is responsible for a delivery of electrons to the anodic electrode. The electrons pass through an electrical circuit, containing a power user. Arriving at the cathodic electrode, they react with the available electron acceptor, such as oxygen. A microbial fuel cell in general is thus able to extract electrical power from the oxidation of bioconvertible substrates (8). Thus far, research on SMFCs primarily involved marine SMFCs and typically resulted in sustained power productions of 9-16 mW m-2 total anodic surface (7, 9, 10). The rice rhizodeposits potentially form substrates for an SMFC. This research aims to exploit this flow of energy by oxidizing the substrates derived from living plants directly at an anode. The purpose is to demonstrate that growing plants can serve as ongoing sources of organic substrates for power generation in a SMFC configuration. To achieve this goal, several SMFC reactors were set up, comprising reactors with and without plants, operated in open and closed electrical circuit and with several types of substratum, namely, soil, vermiculite, and graphite granules.

Materials and Methods Growth of Rice Plants. Rice plants used in the experiments belonged to Oryza sativa ssp. indica, cultivar C101PKT, originating from the International Rice Research Institute. After 1 week humid incubation of the rice seeds at 28 °C and 3 weeks growth in soil or vermiculite, the plants were transplanted into containers equipped with electrodes and further referred to as sediment microbial fuel cell (SMFC) reactors. Nutrients were dosed at a rate of 5 g of (NH4)2SO4 m-2 week-1 and 10 g of FeSO4 · 7H2O m-2 week-1 with soil as support layer, while plants growing in vermiculite were fed with Hoagland’s hydroponics solution (11) every 2 or 3 days. During operation of the SMFC reactors in soil and vermiculite, a regular administering of nutrients was only applied during the first 3 weeks of reactor runs and during 3 weeks around day 150 of the reactor runs. No nutrients were given to the plants in reactors with graphite granules. The omission of nutrients was intended to stimulate root exudation (4). Trials during the Summer of 2006: Construction. For the first two series of SMFC reactors (see Figure 1), plastic containers with an upper area or plant growth area (PGA) of 272.25 cm2 and a total volume of 4.6 L were filled with either VOL. 42, NO. 8, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Schematic representation of the reactor setup. Each of the four separated compartments holds a so-called sediment microbial fuel cell (SMFC): a support matrix in which plants can root and which holds one or more anodes is submerged by water in which a cathode is placed. The SMFC can be planted or not. The support matrix can be, e.g., regular soil, vermiculite, etc. In the case of soil as matrix, the reactors presented above are respectively called soil0 (control reactor in closed circuit, without plants), soil1 (reactor in closed circuit, with plants), soil2 (repetition of soil1), and soilOC (reactor in open circuit, with plants). soil (Structural Professional type 1, M. Snebbout N.V., Kaprijke, Belgium) or vermiculite (exfoliated vermiculite, Sibli SA Vermiculite et Perlite, Andenne, Belgium) as supports for plant growth. These containers were placed in subdivided aquaria filled with tap water to a height of 25 or 8 cm above the support surface, thus leading to a total volume of 13.3 L/(microbial fuel cell). Before placement, two anodic graphite mats (3.18 mm thickness, Alfa Aesar) of 9 cm by 12 cm were placed in the support layers, one at 6 cm and one at 14 cm below the support surface, resulting in a total anodic geometric area (GA) of 216 cm2. Only one anode of 6 cm by 9 cm was placed at a depth of 6 cm below the surface in reactors meant for open circuit (no current harvesting was required). All mats were interwoven with a graphite rod (5 mm diameter, Morgan), which was attached to the electrical circuitry through an insulated connection. The third series of reactors consisted of glass cylinders with a diameter of 14 cm, filled with 11.5 cm of graphite granules (5 mm diameter, Le Carbon), a sand layer of 1.5 cm, and a standing water layer of 5 cm. The anodic structure as a whole had a geometric area, equal to the plant growth area, of 154 cm2. A graphite rod of 13 cm and insulated wire connected the anodic granule matrix to the outside. Four plants grown in soil were planted in the containers filled with soil, while respectively 6 and 2 plants grown in vermiculite were planted in the containers filled with vermiculite and cylinders with granules. Control reactors were unplanted. Bacterial inocula were added to all anodic compartments, by injecting all reactors with 10 mL of the effluent of an acetate oxidizing MFC reactor (12). Soil and vermiculite matrices were furthermore initially mixed with 20 mL of a methanogenic culture (presettling tank of a constructed wetland, Wontergem, Belgium). Two types of cathodes were used. The first type consisted of a tube of cation exchange membrane (Ultrex), closed at the bottom (diameter, 3.4 cm; length, 20 cm). These tubes were filled with graphite granules, a regularly replenished 100 mM phosphate buffered solution of 100 mM K3Fe(CN)6, and a graphite rod for electrical contact. This type of cathode was used for the reactors with graphite granules at the anode (except for granOC, which did not contain a cathode) and during the first 100 and 125 days respectively of the test runs of reactors with soil and vermiculite. During the remaining time of the latter two series of reactors, a biologically catalyzed oxygen reducing cathode was used. This graphite mat cathode (5 cm by 12.5 cm) was connected with a graphite rod, floating 3054

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in the top water layer of the reactors and slightly aerated through an air pump. Before placement of the cathodes, the mats were inoculated with a performant cathodic culture and the oxygen reduction reaction was initiated in separate SMFCs. Trials during the Summer of 2006: Operation. The entire reactor setup consisted of 13 SMFC reactors, 4 with soil (soil), 4 with vermiculite (verm), and 5 with graphite granules (gran) as support material. To denote the different types of reactors, a subscript is added to the support abbreviation: 1 and 2 for the repetitions of the reactors with plants in closed circuit, allowing current generation (soil1, soil2, verm1, verm2, gran1, and gran2), 0 and 0′ for the control reactors without plants in closed circuit (soil0, verm0, gran0, and gran0′) and OC for the reactors with plants in open circuit (soilOC, vermOC, and granOC), where no current is possible. Table S1 of the Supporting Information gives an overview of the different types of reactors and their names. The electrical circuits were closed through the use of a variable external resistance, which at any time had one value (between 75 and 500 Ω) per series of reactors. The reactors were positioned in a greenhouse with a temperature thermostat set at 28 °C and under special horticulture lamps (HQI, 400 W) with a 16-h-day-8-h-night cycle. On the basis of temperature and light conditions in- and outside the greenhouse, the reactor runs can be subdivided in three succeeding test periods, starting from the beginning of April 2006. Average outside shortwave radiation and outside temperatures during test period 1 were 157 W m-2 (second half of this test period) and 13.2 ( 3.5 °C (inside around 30 °C); during test period 2, 231 W m-2 and 20.0 ( 4.0 °C (inside around 42 °C) and during test period 3, 105 W m-2 and 16.1 ( 2.5 °C (with intermediate inside temperatures). Radiation inside the greenhouse was determined to be around 18 MJ m-2 day-1 during the second test period (LICOR, LI-190S, (13)). Test periods 1-3 respectively started on days 1, 52, and 120 of the soil reactor runs, on days 1, 60, and 128 of the verm reactor runs, and days 1, 2, and day 70 of the gran reactor runs. The total reactor runs from the soil and verm reactors respectively comprised 204 and 188 days, thus covering the entire cycle of the rice plant life from transplanting onward. The reactor runs from the gran reactors lasted 142 days. Trials during the Summer of 2007: Construction and Operation. One year after the operation of the three series of reactors described above, a fourth series of reactors was constructed analogous to the soil reactors. This series is referred to as b-soil reactors. The construction of the b-soil reactors differed from that of the soil reactors by the use of plastic containers with an upper PGA of 231.04 cm2, height of 19.6 cm, and total volume of 3.3 L. Three anodic graphite mats (Sigratherm, KFA, 2.5 mm thickness), each of 6 cm by 11 cm, were placed horizontally at respectively 5, 11 and 17 cm below the support surface and resulted in a total anodic geometric area of 198 cm2. The cathode consisted in each case of an aerated graphite mat (Alfa Aesar, 3.18 mm thickness) of 5 cm by 12.5 cm. Three containers were planted with 5 4-week-old rice plants each (b-soil1, b-soil2, and b-soil3), two containers remained unplanted (b-soil0 and b-soil0′) (Table S1). An extra horticulture lamp (HQI, 400 W) was placed above the reactors to account for lower light influx. The reactor runs started in the beginning of June 2007 and could be divided in two test periods. The average shortwave radiation and outside temperature were respectively 166 W m-2 and 18.4 ( 3.6 °C in the first period and 78 W m-2 and 13.6 ( 3.8 °C in the second test period, which started on day 88 of the reactor runs. An external resistance of either 497 or 100 Ω was used. Data collected until day 144 of the reactor run are included in this paper.

Electrochemical Data. Continuous potential measurements were recorded every 5 or 7.5 min. Other electrochemical measurements and the analysis thereof were performed according to Logan et al. (14). Negative signs were assigned to power outputs corresponding with reverse (negative) currents. The expression of current production as chemical oxygen demand (COD) oxidation was based on the release of 4 mol of electrons/(mol of COD oxidized or mol of O2). Chemical Analysis. Chemical oxygen demand analysis was chosen as a general measurement of the amounts of oxidizable material in the solution of the anodic compartment. Mixed samples of the solution were collected with a spinal needle, stored at 4 °C, filtered (Whatman, 5971/2) and analyzed according to the dichromate method (15). The dry weights (DW) of the rice shoots as well as the panicles were determined by drying the rice biomass overnight at 105 °C. The pooled above-ground biomass weights per reactor were divided by the initial number of plants to normalize for the different numbers of plants in reactors with vermiculite and soil. Statistical Analysis. Statistical analysis was performed on the data from Figure 4, excluding the data points from vermOC to prevent biases, resulting in 14 data pairs. On the basis of the shape of the scatterplot of the data, linear, logistic, and nonlinear regressions were applied and the Pearson correlation coefficient (two-tailed) between the transformed COD concentrations and the current productions was determined. A Michaelis–Menten type of model with lateral shift [current ∼ Cmax(COD - X)/(K + (COD - X))] and parameters Cmax (maximum current) ) 2.4, K (Michaelis constant) ) 141, and X (lateral shift) ) 74 resulted in the highest correlation coefficient, being 0.76 (p ) 0.002). Normal distribution of the data sets was checked through P-P plots and the Kolmogorov–Smirnov test. Variability is expressed as standard deviation throughout the paper.

Results Overall Electrochemical Performance. In the summer of 2006, four SMFCssof which three were planted with riceswere set up with natural soil as support and were held in open circuit (no current was allowed) until a cell potential higher than 600 mV was reached, indicating anaerobic conditions around the anode. The stable open circuit cell potential of soilOC, the control reactor with plants that was kept in open circuit throughout the experimental period, slowly increased during the entire reactor run of 204 days from 695 to 800 mV with an anodic redox potential decreasing from -240 mV vs SHE to -340 mV vs SHE. The sharpest increase in open circuit cell potential occurred concomitantly with the start of the second test period, with a stronger sun regime. Notable currents of about 3 mA m-2 anodic geometric area (GA) were harvested from the moment the electrical circuits of reactors soil1 and soil2 (with plants) and soil0 (without plants) were closed. A steady relation between the subsequent, higher level performances of these reactors was only observed from the start of the second test period onward. Figure S1a of the Supporting Information contains a simplified performance profile of the soil reactors through time, representing the cumulative electron transfer. Averaged over the entire period of closed circuit, soil1, soil2, and soil0 respectively delivered 68 ( 45, 54 ( 38, and 40 ( 20 mA m-2 GA. When averaging over concurrent, stable, and representative periods (not limited by the cathode reaction, plant age, or climate), comprising at least 27 days per reactor (between day 63 and day 129), the reactors with plants in soil produced a current of 120 ( 19 mA m-2 GA, which is a factor 2.7 higher than the current of 44 ( 8 mA m-2 GA, produced by the reactor with soil. During that period, the reactors with plants produced a factor 7 more power

FIGURE 2. Electrical power output per m2 geometric anode area (GA) and standard deviation. (a) First three series of reactors, operated in the summer of 2006. At the left side of the figure, power output is given for periods with a ferricyanide cathode (cross-hatched bars) and with an oxygen reducing cathode (black bars). The right side only refers to periods with ferricyanide cathodes. A subscript 0 points to the absence of plants. Each bar depicts a representative performance period per reactor, comprising a period of respectively 16, 15, and 5 days or more in case of reactors with soil (soil), vermiculite (verm), and graphite granules (gran). The cross-hatched bars from soil and vermiculite reactors originated from test period 2; all other bars, from test period 3. (b) Repetition series with soil (b-soil), operated during the summer of 2007. The bars depict representative performance periods of at least 18 days. At any instance, an oxygen reducing cathode was used. A subscript 0 points to the absence of plants. than the control reactor without plants, being 26 ( 7 mW m-2 GA versus 3.7 ( 1.8 mW m-2 GA (Figure 2a; Figure S2 and Table S2 of the Supporting Information). A similar reactor setup was used with vermiculite as support. These SMFCs were held in open circuit for 29 days, during which the obtained open circuit cell potentials remained fluctuating with daily peaks between -50 and +130 mV. At the start of test period 2 (day 60), the cell potentials of the reactors with plants started to increase steeply to reach higher and more stable values: the open circuit cell potential of vermOC, the control reactor with plants that was kept in open circuit throughout the experimental period, reached consistently positive values up to 760 mV, verm1 increased its cell potential with a factor 15 and verm2 changed its regularly negative cell potential into a stable positive cell potential. It required 104 dayssstill in test period 2sfor verm2 to steeply increase its cell potential to become as high as that of verm1. During periods with high cell potentials, a fluctuation in the latter could also be observed, this time without negative cell potentials (Figure 3). These fluctuations were determined by the anodic potentials; high cell potentials and low anodic potentials were reached when light conditions prevailed and low cell potentials and high anodic potentials were reached in the absence of light. The closed circuit reactor without plants (verm0) demonstrated low cell potentials (with a negative average) during the entire reactor run. The VOL. 42, NO. 8, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Fluctuation in voltage produced in reactors containing vermiculite and plants. Vertical bars show the artificial light conditions inside the greenhouse (grey bars ) no light). The external resistance for the reactor in closed circuit was 495 Ω, and a ferricyanide type of cathode was used. Dark line, vermOC; light line, verm1.

FIGURE 4. Anodic organic substrate of the reactors with vermiculite as support layer. Current production, expressed as rate of COD oxidation per day and per liter total anode compartment (TAC), is plotted versus COD concentration measured in the anodic solution. Reactors with plants in closed circuit (b, verm1; O, verm2) and with plants in open circuit (2, vermOC) are presented. cumulative electron transfer of the three reactors with vermiculite support in closed circuit is given in Figure S1b. Although there was variability in the length of the start-up period, the reactors with plants clearly behaved similar to each other and differed from the reactor without plants, which produced low negative currents. The total time-averaged current production of verm1, verm2, and verm0 was respectively 36 ( 17, 14 ( 16, and -0.7 ( 1.0 mA m-2 GA. The mean current averaged over total representative periods of at least 38 days per reactor (between day 54 and day 188) gives 44 ( 9 mA m-2 GA for the reactors with rice (verm1 and verm2) and -0.56 ( 0.69 mA m-2 GA for the reactor without rice plants (verm0). The mean power for verm1 and verm2 was 21 ( 8 mW m-2 GA; that of verm0 was -0.008 ( 0.01 mW m-2 GA (Figure 2a and Table S2). Verm1 could sustain a power production of 33 mW m-2 GA for at least 6 days. A third series of reactors contained graphite granules as plant support. These SMFCs generally yielded low cell potentials, as was the case with the vermiculite reactors. Near the end of the reactor runs, current production from the reactors with plants (gran1 and gran2) started to increase steeply, whereas that of the reactors without plants (gran0 and gran0′) did not. By subtracting the cumulative electron transfer of the last type of reactor from the first one, the net cumulative electron transfer could be obtained (Figure S1c). 3056

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Averaging power production over a high-performance period of a minimum of 5 days per reactor (between day 119 and day 141) yielded 15.8 ( 2.1 mW m-2 GA for the granule reactors with plants and 0.14 ( 0.03 mW m-2 GA for those without plants (Figure 2a). To confirm the test results and show that repetition in time was possible, an additional series of reactors was set up during the summer 1 year later, with soil as support layer (b-soil reactors). The cumulative electron transfer for these reactors is shown in Figure S1d. Averaged over the entire reactor run the reactors b-soil1, b-soil2, b-soil3, b-soil0, and b-soil0′ respectively produced 43 ( 30, 35 ( 27, 39 ( 23, 22 ( 17, and 7 ( 12 mA m-2 GA. The mean current production during a representative period of at least 18 days (between day 62 and day 85) was a factor 3.4 higher for the reactors with plants (b-soil1, b-soil2, and b-soil3) than for those without plants (b-soil0 and b-soil0′), being 68 ( 20 versus 20 ( 12 mA m-2 GA. The mean power output for the reactors with plants was 9.9 ( 6.0 mW m-2 GA, which was a factor 9 higher than the power output from the reactors without plants (1.1 ( 1.1 mW m-2 GA) (Figure 2b; Figure S2 and Table S2). This confirmed the observations of the previous summer. Energy Considerations. The energy obtained through the planted SMFC systems can be compared with the energy cycle of irradiant solar energy, photosynthesis, and exudation. An extended version of these calculations can be found in the Supporting Information. On the basis of the actual rice biomass production in these experiments and a release of 3% as exudates, an exudate release of 0.41 g of C m-2 PGA day-1 was obtained. Considering a general composition of [CH2O] for the released material, this can be converted in a daily release of electrons and energy. The averaged current and power production from the SMFCs, which could be attributed to the plants, was 47.5 and 17 mW m-2 PGA, corresponding with 0.043 mol of electrons and 1.47 kJ m-2 PGA day-1. On the basis of the biomass production and the oxidation of the readily biodegradable theoretical exudate release, the SMFCs with rice plants thus obtain a coulometric efficiency (recovery of electrons) of 31% and an energetic efficiency of 9%. Through a combination of the sunlight interception efficiency (89%; based on LAI), the photosynthetic efficiency for C3 plants (4.9%), the exudates release (3%), and the fuel cell efficiency (9%), an overall efficiency for the conversion of light energy into electrical energy is obtained, being 0.01%. This can be confirmed by comparing the harvested 1.47 kJ m-2 PGA day-1 with the radiation energy in the greenhouse (about 18 MJ m-2 day-1, the same order of magnitude as the solar radiation of 15 MJ m-2 day-1 in rice producing countries). Rice Plants and the Anodic Organic Substrate. Measurements of soluble COD in the interstitial anodic solution of reactors with vermiculite as support were taken as a representative for the amount of reduced organics available as microbial substrate at the anode, as this forms the source of electrical energy in the systems. Higher COD concentrations could be measured in verm1 than verm2. The first reactor generally performed better from an electrical point of view than the latter. There was more COD in solution in reactors producing current than in the reactor left in open circuit (see Figure S3 in the Supporting Information for CODcentrations through time). Figure 4 demonstrates a positive Michaelis–Menten-like correlation between the current production in the vermiculite reactors and the COD concentration in the anodic compartment. This type of correlation might imply that the attainable current output is limited for the used setup. At the end of the reactor runs, the aboveground biomass mainly consisted of senescent leaves, suggesting that decaying plant material could be an important anodic substrate at this point. This was especially the case with the reactors in

graphite granules, where the electrical current production (Figure S1c) notably increased after the plants had almost died off. The reason for this plant mortality presumably was a visible leakage of traces of ferricyanide from the cathode into the anode compartment, since the plants in granOC (open circuit without a ferricyanide cathode) remained healthy (results not shown). The total aboveground production of plant biomass was determined at the end of the soil and verm reactor runs. With an overall production of 27 ( 7 g of DW/(plant in closed circuit) and 21 ( 1 g of DW/(plant in open circuit), there appears to be no effect of the electrical circuit on the plant biomass production.

Discussion In this study it was demonstrated that the presence of plants increased the power production from sediment microbial fuel cells (SMFCs) (see also Figure 2, Figure S2, and Table S2) and that it is possible to oxidize plant-derived material in situ. A reactor with soil as support, holding organic oxidizable substrate, enabled a freshwater SMFC to produce electrical power in a sustained way. The presence of plants increased the current output from this type of reactor with a factor 2.7 and the power output with a factor of 7. The series of 2007, only using soil as support, showed that our data were reproducible during a subsequent summer period. Differences in absolute values and trends between the two soil series are probably related to differences in plant growth and age, time of reactor run, and weather which indeed slightly varied between the two experimental years. If the reactors were filled with a support matrix, which did not contain organic material, being vermiculite (hydroponic plant growth substratum) or graphite granules (anodic matrix for reactor type MFCs), no apparent electrical current could be produced in the absence of plants. The presence of plants, more specifically the presence of living plant roots around the anode, allowed substantial power generation. Roots of living plants transport organic material such as exudates into the rhizosphere. As exudates have a major stimulatory effect on microbial growth and activity because of their rapid assimilation (2), they indeed qualify as a readily oxidizable anodic substrate. The highest sustained electrical output, based on plant-derived material, was an output of 33 mW m-2 GA or 330 W ha-1 GA. On the basis of the oxidation of theoretical exudate release, the SMFCs with rice plants obtained a coulometric efficiency of 31% and an energetic efficiency of 9%. Further engineering improvements on the system are necessary to enable an increase in fuel cell efficiency. The results from Figure 2a demonstrate that the electrical output from a system with ferricyanide at the cathode (used before in reactor type MFCs for a reliable output (12)) was at equivalent levels as that from a system with a sustainable, biologically catalyzed cathode, using dissolved oxygen as cathodic reagent. The electrical current and power produced in the SMFCs in this study can be compared with the power obtained in previous SMFC research, with a similar design but different substrates. A long-term sustained current and power production of 120 mA m-2 GA (geometric anode area) or 56 mA m-2 AS (total anodic electrode surface) and 26 mW m-2 GA or 12 mW m-2 AS was obtained in this work from a rice freshwater ecosystem. Long-term sustained current and power production from marine systems amounted to 34 mA m-2 AS (7) and 16 mW m-2 AS (9). Reimers et al. (16) reached a power output (sustainable for 24 h) of about a factor 2 higher at an ocean seep. Sustained electrical production from a sediment freshwater system amounted to 9 mA m-2 AS (17). Power production is more difficult to obtain from freshwater systems than from salt water systems because of

a lower electric conductivity and decreased reactivity of the electrodes. Hence, the long-term sustained results obtained in this (freshwater) study are substantial. During the trials in 2006, a start-up period of 50-100 days was required in order for the reactors to start producing electrical current derived from living plants (Figure S1). The time delay could be due to many reasons, such as the life cycle dependency of the exudate release, both qualitatively and quantitatively (2, 18), the omission of nutrients (which can induce exudation) (4), the release of oxygen, conducted through the aerenchyma (19), scavenging the electrons otherwise collected at the anode, and the lack of an adapted anodic microbial consortium. Temperature and light conditions are an important factor, as the onset of high voltages of the plant reactors coincided for most reactors with the start of test period 2, with higher temperatures and higher photosynthetic radiation. Higher phototrophic production allows a higher exudate release, while high temperatures have been reported to decrease the oxygen release by plants (20) as well as increase the exudate release (2). The vermiculite reactor without plants was not affected by the periods of high ambient temperature. Temperature and photosynthetic radiation were higher at the onset of the trials in 2007 than at the onset in 2006, which could explain the earlier onset of stable trends in 2007. The fluctuations in cell potential of reactors with plants, determined by the anodic potentials (as the cathodic potentials were relatively stable, Figure 3), were presumably due to the presence of reduced, oxidizable compounds around the anode. The release of the latter is higher during the day, as demonstrated by Leake et al. (3). A relatively higher concentration of reduced compounds would result in a lower anodic potential and hence a higher cell potential during the day. Photosynthesis thus determines the cell potential of the fuel cells and the power that can be extracted from these reactors. The positive relationship between COD and current production (Figure 4) demonstrates that the COD derived from the plants (representing rhizodeposition) acts as an electron donor for electricity generation. The presence of an oxidizing anode leads to higher concentrations of COD (Figure 4). The latter infers that the withdrawal of rhizodeposits appears to stimulate a further excretion of reduced substrates by the roots. This is in accordance with the findings of Barber and Lynch (21), who found an increased exudation in the presence of microorganisms, possibly through the utilization of exudates. Attempts to characterize the substances comprising the measured COD are warranted. The rice plants studied in our reactors produced current during thousands of hours of their plant life. A large part of this was during an active growth period, suggesting that excreted compounds, mucilage, and sloughed-off cells, etc. made up the anodic substrate, aside from the oxidation of dead plant material. The latter would notably occur at the end of the reactor runs, when the aboveground biomass mainly consisted of senescent leaves. The fact that current can be obtained from the oxidation of decomposing root residues is in accordance with the observation that root residues are the major contributor to methane production (5). Photosynthetic activity has been applied before to drive the electricity generation in reactor type MFCs, involving whole-cell photosynthetic microorganisms or subcellular photosynthetic components and requiring either electrocatalyzed anodes or the addition of redox mediators (22, 23). This paper demonstrated for the first time that living higher plants can provide an ongoing supply for electrical current production in a sediment type of microbial fuel cell, without the need for chemical catalysts or added redox mediators. The anodic oxidation of organic compounds set free by plant roots into the rhizosphere offers several environmental perspectives. A rice paddy field or any vegetated wetland VOL. 42, NO. 8, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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system could thus for instance give rise to a renewable resource for direct electricity production by oxidizing a substrate, which is conventionally transformed into methane. A potential is offered for a sun-driven power generation in (remote) areas with high solar inputs without the need for scarce and costly construction materials such as silicon used to fabricate photovoltaic cells. This nondestructive energy production from biomass occurs without the need of costly and/or energy consuming conversion techniques. Whereas a typical sediment MFC is furthermore limited by diffusion to the anode, a living plant can continuously deliver substrate close to the anode, allowing continuous power production and increasing the attainable production from a typical nonplanted SMFC. Alternatively, this type of SMFC might be applied to influence redox-dependent processes in the rhizosphere. For example, through the presence of the oxidizing anode, an electron acceptor for respiration pathways is continuously available in the rhizosphere, allowing a full oxidation of the plant substrates. The technique could hence offer the prospect to mitigate the sediment redox potential and abate undesirable processes such as methylation of metals and emission of methane, which will be investigated in subsequent experiments.

Acknowledgments L.D.S. is supported through a Ph.D. grant from the Bijzonder Onderzoeks Fonds of Ghent University (Grant No. 01D24405). K.R. is supported by the UQ Postdoctoral Research Fellow scheme and the ARC Discovery program. The supply of rice seeds and advice on rice growth from David De Vleesschauwer were greatly appreciated. The useful comments of Peter Aelterman, Peter Clauwaert, Hai The Pham, Jorge Sanchez Martinez, Michael Friedrich, and Angela Cabezas are kindly acknowledged. The supply of a PAR sensor and climatic data by the Laboratory of Plant Ecology and the Laboratory of Wood Technology, Ghent University, was highly appreciated.

Supporting Information Available Table S1 containing an overview of the rice reactor names and configurations, Table S2 giving an overview of reactor performances), Figure S1 showing cumulative electron transfer, Figure S2 illustrating power ratios, Figure S3 picturing anodic substrate, and an extended version of the energy considerations from Results and additional literature. This material is available free of charge via the Internet at http://pubs.acs.org.

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