Electricity Production Coupled to Ammonium in a ... - ACS Publications

Mar 23, 2009 - addition. Using 16S rRNA-linked molecular analyses, we found ... pounds in wastewater or agricultural wastes (1). The overload .... DGG...
1 downloads 0 Views 2MB Size
Download PDF
Environ. Sci. Technol. 2009, 43, 3391–3397

Electricity Production Coupled to Ammonium in a Microbial Fuel Cell Z H E N H E , †,‡ J I N J U N K A N , † YANBING WANG,† YUELONG HUANG,‡ FLORIAN MANSFELD,‡ AND K E N N E T H H . N E A L S O N * ,† Department of Earth Sciences, University of Southern California, Los Angeles, California 90089, and Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089

Received December 9, 2008. Revised manuscript received March 2, 2009. Accepted March 9, 2009.

The production of electricity from ammonium was examined using a rotating-cathode microbial fuel cell (MFC). The addition of ammonium chloride, ammonium sulfate, or ammonium phosphate (monobasic) resulted in electricity generation, while adding sodium chloride, nitrate, or nitrite did not cause any increase in current production. The peak current increased with increasing amount of ammonium addition up to 62.3 mM of ammonium chloride, suggesting that ammonium was involved in electricity generation either directly as the anodic fuel or indirectly as substrates for nitrifiers to produce organic compounds for heterotrophs. Adding nitrate or nitrite with ammonium increased current production compared to solely ammonium addition. Using 16S rRNA-linked molecular analyses, we found ammonium-oxidizing bacteria and denitrifying bacteria on both the anode and cathode electrodes, whereas no anammox bacteria were detected. The dominant ammonium-oxidizing bacteria were closely related to Nitrosomonas europaea. The present MFC achieved an ammonium removal efficiency of 49.2 ( 5.9 or 69.7 ( 3.6%, depending on hydraulic retention time, but exhibited a very low Coulombic efficiency.

Introduction Ammonium is one of the most important inorganic compounds in wastewater or agricultural wastes (1). The overload of ammonium with other nutrients discharged from wastewater, fertilizers, and human activities can result in eutrophication that deteriorates water quality and aquatic ecosystems (2). Ammonium in wastewater is usually converted into dinitrogen gas via nitrification/denitrification or anammox (anaerobic ammonium oxidation) (3). In general, it would be beneficial if useful energy could be generated via the harvesting of the chemical energy in ammonium during its oxidative removal. One promising approach to realize this goal is ammonium-based microbial fuel cells (MFCs), producing electricity in the same way that such systems have been employed to produce electric power from the oxidation of various organic or inorganic compounds (4-6). However, to date, the production of electricity from ammonium * Corresponding author phone: (213) 821-2271; fax: (213) 7408801; e-mail: [email protected]. † Department of Earth Sciences, University of Southern California. ‡ Mork Family Department of Chemical Engineering and Materials Science, University of Southern California. 10.1021/es803492c CCC: $40.75

Published on Web 03/23/2009

 2009 American Chemical Society

oxidation in MFCs has not been reported. In one study, high levels of ammonium were removed in a MFC used to treat swine wastewater (7), but further investigation concluded that ammonium was not a substrate for electricity generation, and its removal was largely due to either ammonium volatilization in an air-cathode MFC or ammonium ion diffusion from the anode to the cathode in a two-chambered MFC (8). Ammonium may contribute to electricity generation in MFCs by two ways. First, because ammonium-N is at its lowest oxidation state and can supply electrons via its oxidation, ammonium may function as an anodic fuel in MFCs. Ammonium oxidation occurs under aerobic and anaerobic conditions, both of which exhibit negative Gibbs free energy (under standard conditions) of -357 (anaerobic) and -275 KJ/mol (aerobic) (9). As a result, the standard potentials of both reactions are positive, indicating that they can theoretically generate electric energy in MFCs with ammonium as an electron donor (anode) and nitrite/nitrate or oxygen as an electron acceptor (cathode). Second, ammonium may be utilized by nitrifying bacteria to produce organic compounds that are used by heterotrophs to generate electricity. It is known that autotrophic nitrifying bacteria can support heterotrophic growth by producing soluble microbial products (10). Although the mechanistic details of ammonium oxidation remain unsolved, we report here the ammonium-dependent generation of electricity. The results indicate that (1) the addition of ammonium to organic-free MFCs could generate electricity; (2) a coupled aerobic/anaerobic set of reactions appeared to be operating populations at both the anode and cathode; and (3) a variety of microbes, dominated by nitrifying bacteria and denitrifying bacteria, were present at both of the electrodes of the MFC.

Materials and Methods Rotating-Cathode MFC Setup. The rotating-cathode MFC (Figure 1) was constructed according to a previous study (11). The anode electrode, a piece of graphite plate (6 × 25 cm2) (POCO Graphite Inc., Decatur, TX), was placed on the bottom of a rectangular plastic chamber (25[L] × 7.5[H] × 6[W] cm3, with a liquid load of ∼750 mL). The cathode electrodes consisted of 10 pieces of round graphite felt (Electrolytica Inc., Amherst, NY) with a diameter of 5.5 cm. The graphite felts were connected in series by a graphite rod (POCO Graphite Inc.) that was attached to a pump drive (Cole-Parmer Instrument Company, Vernon Hill, IL). The cathode electrodes were installed above the anode electrode so that ∼40% of the graphite felt was immersed in the solution. The distance between the bottom of the graphite felts and the top of the anode electrode was about 2.5 cm. The MFC was shielded from light to prevent phototrophic reactions. A 1000 Ω resistor was connected between the anode and cathode electrodes using copper wire. No membrane was placed between them. Operating Conditions. The MFC was operated at a room temperature (23-25 °C) as a sequencing batch reactor (SBR), an approach already proven to be an effective method for the cultivation of slowly growing anaerobic ammoniumoxidizing bacteria (12). A mixture of aerobic and anaerobic sludge (mixing ratio 1:1) from a wastewater treatment plant (the Joint Water Pollution Plant, CA) was used to inoculate the MFC and allowed to sit for 2 days before the daily flushing began. The nutrient solution contained (per L of tap water) NaHCO3, 1.5 g; MgSO4, 0.002 g; CaCl2, 0.015 g; KH2PO4, 0.005 g; K2HPO4, 0.01 g; and trace element, 1 mL (13). The pH of VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3391

FIGURE 1. (A) Schematic and (B) picture of laboratory prototype rotating-cathode MFC. The anode electrode was a piece of graphite plate placed on the bottom of a rectangular plastic chamber. The cathode electrodes consisted of 10 pieces of round graphite felt. The MFC was flushed once each day, giving an HRT of 1 day. the nutrient solution was monitored but not adjusted. One liter of the nutrient solution was pumped through the MFC in a period of 1 h once a day, ensuring a maximum removal of the residues from the previous day. On the basis of the rapid return of current production, the anode biomass was assumed to be maintained in a healthy state during the flushing. Thus, the hydraulic retention time (HRT) was 1 day, except for a few tests with a longer HRT of 6 days (one flush every 6 days). Ammonium chloride (or other ammonium compounds) was added into the MFC using a spoon immediately after the nutrient solution flushing stopped. Ammonium chemicals dissolved and were distributed slowly by the agitation of the cathode electrode rotation at a speed of ∼1.1 rpm. Electrochemical and Chemical Measurement. The cell voltage was recorded every 30 s by a digital multimeter (2700, Keithley Instruments, Inc., Cleveland, OH). The concentration of ammonium-N was determined using an ammonium ion electrode according to the procedure provided by the manufacturer (Cole-Parmer Instrument Company, Vernon Hills, IL). Nitrite and nitrate were measured spectrophotometrically (14). The pH was measured using a Benchtop pH meter (UB-10, Denver Instrument, Denver, CO). Coulombic efficiency was calculated by dividing coulomb output (integrating current and time) by total coulomb input (based on inorganic ammonium) according to previous literature (13). Sample Collection and DNA Extraction. A slice of cathode electrode, 0.3 g of sediment (anode), and original inoculum (0.3 g of mixed sludge) were collected and genomic DNA was extracted by UltraClean Soil DNA kit (MO BIO Laboratories, Carlsbad, CA) following the manufacturer’s instructions. DNA concentration was estimated on the basis of 260 nm 3392

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 9, 2009

absorbance using a Spectrophotometer ND-1000 (NanoDrop Products, Wilmington, DE). PCR and DGGE. PCR amplification was performed in a 50 µL reaction containing approximately 25 ng of template DNA, 25 µL of PCR Mastermix (Qiagen), 0.5 mM (each) primer, and dd water. PCR program was performed with a Mastercycler gradient (Eppendorf, Hamburg, Germany). PCR primers used were 341f (GC) and 907r and the PCR program followed the protocol described by Sca¨fer and Muyzer (15). Agarose gel electrophoresis was used to detect and estimate the concentrations of PCR amplicons. DGGE was performed as previously described (16) except the linear gradient of the denaturants was from 40-70% instead of 40-65%. Detection of Anammox Bacteria. Anammox bacteria are members of the Planktomycetes phylum. To detect anammox bacteria on both the anode and cathode electrodes, a sequential PCR approach was employed. First, Planctomycetales-specific 16S rRNA gene was amplified by using the Pla46 primer (17) and universal primer 1392r (18). Following this, amplification of the 16S rRNA gene for DGGE was performed using universal bacteria-specific primers 1070f and GC clamp primer 1392r (18). DGGE Band Sequencing and Phylogenetic Analysis. Representative bands were excised from DGGE gels and incubated in diffusion buffer (0.25 M ammonium acetate, 10 mM magnesium chloride, and 0.1% SDS) at 50 °C for 30 min. One microliter of supernatant was used to reamplify the band. PCR products were purified by ExoSAP-IT (USB, Cleveland, OH) and sequenced with primer 341f (no GC) by using Bigdyeterminator chemistry by ABI PRISM3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). All sequences were compared with GenBank database using BLAST, and the closest matched sequences were obtained and included in

the downstream analysis. Phylogenetic trees were constructed using MacVector 10.0 software package (MacVector Inc., Cary, NC). Briefly, sequence alignment was performed with the program CLUSTAL W. Evolutionary distances were calculated using the Jukes-Cantor method (19) and distance trees were constructed using the neighbor-joining algorithm (20). Bootstrap values were obtained on the basis of the analysis of 1000 resampling data sets. Sequences of the partial 16S rRNA genes of representative DGGE bands have been deposited in the GenBank database under accession numbers FJ418973-FJ418983.

Results Electricity Generation from Ammonium. After 2 months’ operation, the electric current generation began, and has continued for more than 5 months (data not shown). To exclude the possibility that increasing ionic strength was responsible for the current production, we added nonammonium-containing salts, including NaCl, NaNO3, and KNO2, none of which stimulated current production (Figure 2A). The rapid drop in current production occurred as the fresh nutrient solution was flushed through the MFC. When 24.9 mM NH4Cl was added, the current gradually increased from 0 to 0.062 mA in 12 h. The second addition of 24.9 mM NH4Cl generated a peak current of 0.068 mA. The same addition was repeated multiple times and the MFC produced a peak current between 0.057 and 0.062 mA (Figure 2B). Other ammonium-containing salts (NH4H2PO4 and (NH4)2SO4) also produced electricity, and the peak currents were around 0.046 mA when the amount added was adjusted to the same molarities as used for the NH4Cl (Figure 2C). Current production was shown to be dependent on the concentration of ammonium up to 74.7 mM (panels A and B in Figure 3). Tests were done in triplicate to confirm these results (Figure 3B). The highest peak current of 0.078 ( 0.003 mA occurred at an addition of 62.3 mM NH4Cl. Effect of Nitrate or Nitrite on Current Generation. Adding nitrate or nitrite alone had no effect on current production, but when either nitrate or nitrite was added in addition to ammonium, an enhancement of current production was seen (Figure 4). Compared with NH4Cl addition alone (∼ 0.055 mA), the peak current increased to 0.066 mA with the addition of 15.7 mM NaNO3 and 24.9 mM NH4Cl (arrow c). When 15.7 mM KNO2 was added with 24.9 mM NH4Cl (arrow e), the peak current rose to 0.081 mA, which was further increased to 0.092 mA at the addition of 31.4 mM KNO2 and 24.9 mM NH4Cl (arrow f). Replicate tests confirmed the stimulation by both nitrate and nitrite when added with ammonium (Figure 4B). Ammonium Removal. Ammonium concentration was monitored after the addition of 24.9 mM of NH4Cl. At a HRT of 1 day, 49.2 ( 5.9% of ammonium was removed after one day’s operation. The main product was nitrite, which accounted for 69.3 ( 9.9% of ammonium removal, whereas nitrate production accounted for only 14.4 ( 19.9% of ammonium removal. Because the present MFC is an open system, ammonia volatilization would play an important role in ammonium removal, although the low electrolyte pH was observed in this study, which was not favorable for forming ammonia gas (Figure 5). At 25 °C, about 4-9% aqueous ammonium was in the form of NH3 at the pH of the fresh nutrient solution (7.97 ( 0.18), whereas the pH rapidly dropped (Figure 5) and after one day’s operation almost no NH3 was present at the pH of 5.72 ( 0.07 (21). The Coulombic efficiency (CE) calculated for a single addition of NH4Cl and a HRT of 1 day was only 0.06 ( 0.00%. When the HRT was extended to 6 days, the ammonium removal was 69.7 ( 3.6% and the CE was increased to 0.34 ( 0.02%. Analyses of Bacterial Communities. The molecular (DGGE) analysis revealed that the bacteria on electrodes were

FIGURE 2. Electricity generation from ammonium: (A) addition of 22.8 mM NaCl, 15.7 mM NaNO3, 15.7 mM KNO2, and 24.9 mM NH4Cl; (B) repeated addition of 24.9 mM NH4Cl; (C) addition of 25 mM NH4H2POb, 12.2 mM (NH4)2SO4, and 24.9 mM NH4Cl. Arrows indicate the replenishment of the nutrient solution and addition of chemicals. different from the bacterial composition of the original inoculum, whereas similar DGGE band patterns were observed from the anode and cathode microbes (Figure 6). The original inoculum mainly contained Betaproteobacteria (bands 12 and 14) and Firmicutes (band 13). However, ammonium-oxidizing and denitrifying bacteria dominated in both anode and cathode bacterial assemblages. For instance, DGGE bands 1 and 2 were affiliated with the ammonium-oxidizing Betaproteobacteria, Nitrosomonas europaea, whereas bands 3, 5, and 7 were closely related to the autotrophic denitrifying bacteria Comamonas sp. IA-30. Bands 4 and 6 showed high similarity with the denitrifying bacteria isolated from activated sludge (Diaphorobacter nitroreducens), and bands 10 and 11 were similar to a denitrifying Gammproteobacterium, Bacterium CYCU-0215. No evidence for the presence of anammox bacteria was seen at either the anode or the cathode. VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3393

FIGURE 3. Effect of ammonium dosage on current generation: (A) current production at a series of ammonium dosage (from 0 to 74.7 mM NH4Cl); (B) the mean values of peak current at different ammonium dosage. Arrows indicate the replenishment of the nutrient solution and addition of NH4Cl. Error bars represent standard deviation based on triplicate measurements from a single reactor.

Discussion The experimental results indicate that electric current production is coupled to ammonium addition, and that ammonium functions as the substrate either directly or indirectly for electricity generation (Figure 2 and 3). To the best of our knowledge, this is the first experimental evidence of ammonium-dependent electricity production in MFCs. Given that no current was produced for the first 2 months, we assume that abiotic ammonium oxidation and current production are not occurring, and that the current production observed was the result of the enrichment of an ammonium oxidizing community capable of generating electric current. These results are different from those presented in a recent publication, which found no evidence for electricity production via ammonium oxidation (8). We believe our study has incorporated several features that may help to develop an ammonium-based MFC. First, chemolithoautotrophic ammonium-oxidizing microorganisms may not be able to compete with heterotrophic organisms in the presence of organic compounds (22). Accordingly, our ammonium-fed MFCs were enriched under conditions designed for wastewater that contains much ammonium and minimal levels of organic carbon (e.g., the effluent from secondary treatment without nitrification/denitrification process). Second, it may require a long start-up time to develop a microbial community as being demonstrated by the time course of current production in this study. In the early stages of enrichment, intermittent injection of ammonium for 1-2 days would not 3394

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 9, 2009

FIGURE 4. (A) Current production from the addition of nitrate or nitrite with ammonium: (a) 24.9 mM NH4Cl, (b) 24.9 mM NH4Cl, (c) 24.9 mM NH4Cl + 15.7 mM NaNO3, (d) 24.9 mM g NH4Cl, (e) 24.9 mM g NH4Cl + 15.7 mM KNO2, (f) 24.9 mM g NH4Cl + 31.4 mM KNO2. (B) Mean values of peak currents. Arrows indicate the replenishment of the nutrient solution and addition of chemicals. Error bars represent standard deviation based on triplicate measurements from a single reactor.

FIGURE 5. Current production (solid line) and pH change (dark square) are presented as a function of time. The arrow indicates the replenishment of the basic feed solution and addition of 24.9 mM of NH4Cl. be expected to stimulate current generation because of a lack of appropriate microbial community. Third, although the current levels in this study are low, the processes reported here show the potential for using MFC-like systems for both ammonium removal and electricity production. In the absence of nitrite, this process requires oxygen to convert some ammonium (partial oxidation) into nitrite (23). A

FIGURE 6. DGGE gel image of bacteria on cathode (C), anode (A), and original inoculum (I), and phylogenetic construction of DGGE band sequences. complete anaerobic condition would not help to cultivate microorganisms that conduct the process in MFCs, unless nitrite is externally added. Nitrate and/or nitrite play a role in electricity generation, likely as electron acceptors, indicated by the increase of current generation when adding nitrate or nitrite with ammonium. The increased ionic strength due to the addition of nitrate or nitrite may also contribute to current increase (24). However, when the same amount of nitrate or nitrite was added with ammonium, nitrite resulted in 19.7% higher current generation than nitrate (Figure 4), indicating that nitrite or nitrate functioned more than increasing ionic concentration. Indeed, previous studies have reported that both nitrate and nitrite can function as electron acceptors in the cathode of MFCs (25, 26). DGGE analyses suggested that diverse denitrifying bacteria were enriched on both electrodes. Among them, bands 10 and 11 were similar to Gammaproteobacterium CYCU-0215, which was isolated from an activated sludge system and identified as a major denitrifying bacteria reducing nitrite (27). In addition, Comamonas sp. and D. nitroreducens (bands 3-7) belong to the family Comamonadaceae, Betaproteobacteria, and have been recently found in denitrifying bacterial communities (28). Recent studies by characterization of nitrite reductase and nitrous oxide reductase further demonstrated that members of the Betaproteobacteria, especially those of the family Comamonadaceae actually played the primary roles in the denitrification process (29). This study has not yet provided definite information on the actual functions of

these bacteria in MFCs. However, on the basis of the results obtained here, it is logical to hypothesize that denitrifying bacteria work together with ammonium-oxidizing bacteria to facilitate electron transport from ammonium to nitrite, and thus generate electric current. The fact that nitrate addition caused less current increase than nitrite (containing the same nitrogen equivalent) is possibly due to the fact that nitrite, instead of nitrate, is the terminal electron acceptor. Therefore, the current production via nitrate and ammonium depends on nitrate reduction (nitrite production). This is in accordance with the finding that during ammonium oxidation with nitrate, it is believed that nitrite, the product of nitrate reduction, is the oxidant for ammonium (30). On the basis of these findings, an aerobic/anaerobic ammonium oxidation process is proposed to illustrate electron transfer in this rotating-cathode MFC. Because of the presence of oxygen (both the anode and cathode potentials were above 0 V vs Ag/AgCl, indicating the oxygen intrusion into the system) and the availability of ammonium to both the anode and cathode electrodes, aerobic ammonium oxidation is expected to be a dominant process. This is consistent with the result that the ammoniumoxidizing bacterium, Nitrosomonas europaea, was found dominant on both the anode and cathode electrodes. N. europaea derives energy for growth from the oxidation of ammonium by the successive function of ammonium monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) (31). To this end, Schmidt and Bock (32) have demonstrated that N. europaea is able to anaerobically VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3395

oxidize ammonium using nitrite as the oxidant. It is proposed here that on the anode electrode, the majority of N. europaea perform aerobic ammonium oxidation, consume oxygen, and create an anoxic environment for the minor population of N. europaea to conduct anaerobic ammonium oxidation that transfers electrons to the anode electrode. The presence of denitrifying bacteria on the anode electrode accelerates ammonium oxidation by taking up nitrite. Likewise, microanaerobic condition is built up inside the cathode electrode through the consumption of oxygen by aerobic Biofilm outside (see Figure S1 in the Supporting Information). As a result, denitrifying bacteria reduce nitrite, the product of aerobic ammonium oxidation, partially by accepting electrons from the cathode electrode. We can not exclude the possibility that during ammonium oxidation, autotrophs produced organic compounds for heterotrophs to generate electricity. The present study solely emphasizes the feasibility of electricity generation from ammonium addition. The rotating-cathode MFC in this study is not an efficient system to convert ammonium into electricity, as demonstrated by the low CEs. The CEs can certainly be further increased at a lower external resistor (a higher current), but it was not the focus of this study. The accumulation of nitrite indicates that ammonium removal is mainly due to partial nitrification. It is believed that the lack of an anaerobic condition adjacent to the anode, possibly because of oxygen intrusion via cathode rotation (11), accounts for the inefficient electricity production. The design of MFCs to create better anaerobic conditions for the anode is currently in process. Nevertheless, this study has provided a proof of concept that ammonium can contribute to generate electricity in MFCs. It has important implications for the future application of MFC technology in wastewater treatment. An efficient ammonium-fed MFC may be employed to remove ammonium-N from wastewater and agricultural wastes under carbon-limited conditions. To achieve complete ammonium removal in MFCs, a pretreatment step that converts partial ammonium to nitrite may be required, similar to that in an anammox process (33). However, compared with anammox, the ammonium-fed MFC generates electricity as a surplus benefit and thus is more favorable for energyconsuming wastewater treatment. Work is now underway to advance this technology with improvements of both Coulombic efficiency and power output.

Acknowledgments We thank Lewis Hsu (University of Southern California) for providing wastewater sludge as inocula. We also thank anonymous reviewers for helpful comments.

Supporting Information Available SEM pictures of Biofilm on the cathode electrode (PDF). This material is available free of charge via the Internet at http:// pubs.acs.org.

Literature Cited (1) Wiesmann, U. Biological nitrogen removal from wastewater. In Advances in Biochemical Engineering/Biotechnology; Fiechter, A., Ed.; Springer: Berlin, 1994; Vol. 51, pp 113-153. (2) Camargo, J. A.; Alonso, A. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: A global assessment. Environ. Int. 2006, 32, 831–849. (3) Schmidt, I.; Sliekers, O.; Schmid, M.; Bock, E.; Fuerst, J.; Kuenen, J. G.; Jetten, M. S. M.; Strous, M. New concepts of microbial treatment processes for the nitrogen removal in wastewater. FEMS Microbiol. Rev. 2003, 27, 481–492. (4) Angenent, L. T.; Karim, K.; Al-Dahhan, M. H.; Wrenn, B. A.; Domiguez-Espinosa, R. Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends Biotechnol. 2004, 22, 477–485. 3396

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 9, 2009

(5) Rabaey, K.; Verstraete, W. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol. 2005, 23, 291– 298. (6) Lovley, D. R. Bug juice: harvesting electricity with microorganisms. Nat. Rev. Microbiol. 2006, 4, 497–508. (7) Min, B.; Kim, J.; Oh, S.; Regan, J. M.; Logan, B. E. Electricity generation from swine wastewater using microbial fuel cells. Water Res. 2005, 39, 4961–4968. (8) Kim, J. R.; Zuo, Y.; Regan, J. M.; Logan, B. E. Analysis of ammonia loss mechanisms in microbial fuel cells treating animal wastewater. Biotechnol. Bioeng. 2008, 99, 1120–1127. (9) Jetten, M. S. M.; Wagner, M.; Fuerst, J.; van Loosdrecht, M.; Kuenen, G.; Strous, M. Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process. Curr. Opin. Biotechnol. 2001, 12, 283–288. (10) Kindaichi, T.; Ito, T.; Okabe, S. Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization. Appl. Environ. Microbiol. 2004, 70, 1641–1650. (11) He, Z.; Shao, H.; Angenent, L. T. Increased power production from a sediment microbial fuel cell with a rotating cathode. Biosens. Bioelectron. 2007, 22, 3252–3255. (12) Strous, M.; Heijnen, J. J.; Kuenen, J. G.; Jetten, M. S. M. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 1998, 50, 589–596. (13) He, Z.; Minteer, S. D.; Angenent, L. T. Electricity generation from artificial wastewater using an upflow microbial fuel cell. Environ. Sci. Technol. 2005, 39, 5262–5267. (14) Doane, T. A.; Horwath, W. R. Spectrophotometric determination of nitrate with a single reagent. Anal. Lett. 2003, 36, 2713–2722. (15) Scafer, H.; Muyzer, G. Denaturing gradient gel electrophoresis in marine microbial ecology. In Methods in Microbiology; Paul, J., Ed.; Academic Press: London, 2001; pp 425-468. (16) Kan, J. J.; Wang, K.; Chen, F. Temporal variation and detection limit of an estuarine bacterioplankton community analyzed by denaturing gradient gel electrophoresis (DGGE). Aquat. Microb. Ecol. 2006, 42, 7–18. (17) Neef, A.; Amann, R.; Schlesner, H.; Schleifer, K. H. Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes. Microbiology (Reading, U.K.) 1998, 144, 3257–3266. (18) Ferris, M. J.; Muyzer, G.; Ward, D. M. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl. Environ. Microbiol. 1996, 62, 340–346. (19) Jukes, T. H.; Cantor, C. R. Evolution of protein molecules. In Mammalian Protein Metabolism; Munro, H. N., Ed.; Academic Press: New York, 1969; pp 21-132. (20) Saitou, N.; Nei, M. The Neighbor-Joining Method-a New Method for Reconstructing Phylogenetic Trees. Mol. Biol. Evol. 1987, 4, 406–425. (21) Emerson, K.; Russo, R. C.; Lund, R. E.; Thurston, R. V. Aqueous Ammonia Equilibrium Calculations - Effect of Ph and Temperature. J. Fish. Res. Board Can. 1975, 32, 2379–2383. (22) Verhagen, F. J. M.; Laanbroek, H. J. Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Dual EnergyLimited Chemostats. Appl. Environ. Microbiol. 1991, 57, 3255– 3263. (23) Sliekers, A. O.; Derwort, N.; Gomez, J. L. C.; Strous, M.; Kuenen, J. G.; Jetten, M. S. M. Completely autotrophic nitrogen removal over nitrite in one single reactor. Water Res. 2002, 36, 2475– 2482. (24) Liu, H.; Cheng, S.; Logan, B. E. Power generation in fed-batch microbial fuel cells as a function of ionic strength, temperature, and reactor configuration. Environ. Sci. Technol. 2005, 39, 5488– 5493. (25) Clauwaert, P.; Rabaey, K.; Aelterman, P.; Schamphelaire, L. D.; Pham, T. H.; Boeckx, P.; Boon, N.; Verstraete, W. Biological denitrification in microbial fuel cells. Environ. Sci. Technol. 2007, 41, 3354–3360. (26) Virdis, B.; Rabaey, K.; Yuan, Z.; Keller, J. Microbial fuel cells for simultaneous carbon and nitrogen removal. Water Res. 2008, 42, 3013–3024. (27) You, S. J. Identification of denitrifying bacteria diversity in an activated sludge system by using nitrite reductase genes. Biotechnol. Lett. 2005, 27, 1477–1482. (28) Khan, S. T.; Horiba, Y.; Yamamoto, M.; Hiraishi, A. Members of the family Comamonadaceae as primary poly(3-hydroxybu-

tyrate-co-3-hydroxyvalerate)-degrading denitrifiers in activated sludge as revealed by a polyphasic approach. Appl. Environ. Microbiol. 2002, 68, 3206–3214. (29) Khan, S. T.; Horiba, Y.; Takahashi, N.; Hiraishi, A. Activity and community composition of denitrifying bacteria in poly(3hydroxybutyrate-co-3-hydroxyvalerate)-using solid-phase denitrification processes. Microbes Environ. 2007, 22, 20–31. (30) Thamdrup, B.; Dalsgaard, T. Production of N-2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl. Environ. Microbiol. 2002, 68, 1312– 1318. (31) Chain, P.; Lamerdin, J.; Larimer, F.; Regala, W.; Lao, V.; Land, M.; Hauser, L.; Hooper, A.; Klotz, M.; Norton, J.; Sayavedra-

Soto, L.; Arciero, D.; Hommes, N.; Whittaker, M.; Arp, D. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 2003, 185, 2759–2773. (32) Schmidt, I.; Bock, E. Anaerobic ammonia oxidation by cell-free extracts of Nitrosomonas eutropha. Antonie van Leeuwenhoek 1998, 73, 271–278. (33) van Dongen, U.; Jetten, M. S. M.; van Loosdrecht, M. C. M. The SHARON((R))-Anammox((R)) process for treatment of ammonium rich wastewater. Water Sci. Technol. 2001, 44, 153–160.

ES803492C

VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3397