Microbial reduction of nitrate in the presence of zero-valent iron and biochar

Bioresource Technology 200 (2016) 891–896

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Microbial reduction of nitrate in the presence of zero-valent iron and biochar Seok-Young Oh a,⇑, Yong-Deuk Seo a, Beomseok Kim b, In Young Kim b, Daniel K. Cha b a b

Department of Civil and Environmental Engineering, University of Ulsan, Ulsan 44610, South Korea Department of Civil and Environmental Engineering, University of Delaware, Newark, DE 19716, USA

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Biochar enhances the anaerobic

denitrification of nitrate by microbes with Fe(0).  Electron conductivity of biochar accounts for the enhancement.  Increasing dosage of mixed culture promotes nitrate reduction with Fe(0) and biochar.  A product of microbial reduction with Fe(0) and biochar is N2.  A biochar–Fe(0)–microbes system may be an alternative option for NO3 treatment.

a r t i c l e

i n f o

Article history: Received 7 October 2015 Received in revised form 8 November 2015 Accepted 9 November 2015 Available online 14 November 2015 Keywords: Nitrate Anaerobic denitrification Biochar Zero-valent iron Microbes

a b s t r a c t The denitrification of nitrate (NO3 ) by mixed cultures in the presence of zero-valent iron [Fe(0)] and biochar was investigated through a series of batch experiments. It was hypothesized that biochar may provide microbes with additional electrons to enhance the anaerobic biotransformation of nitrate in the presence of Fe(0) by facilitating electron transfer. When compared to the anaerobic transformation of nitrate by microbes in the presence of Fe(0) alone, the presence of biochar significantly enhanced anaerobic denitrification by microbes with Fe(0). Graphite also promoted the anaerobic microbial transformation of nitrate with Fe(0), and it was speculated that electron-conducting graphene moieties were responsible for the improvement. The results obtained in this work suggest that nitrate can be effectively denitrified by microbes with Fe(0) and biochar in natural and engineered systems. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Nitrate (NO3 ), a well-known contaminant in surface water and groundwater, has been extensively studied for its treatment in natural and engineered systems over the last few decades (Ghafari et al., 2008). Nitrate can be discharged to the environment from a variety of sources, including agricultural activity, animal waste, ⇑ Corresponding author at: Department of Civil and Environmental Engineering, University of Ulsan, 93 Daehak-ro, Nam-gu, Ulsan 44610, South Korea. Tel.: +82 52 259 2752; fax: +82 52 259 2629. E-mail address: [email protected] (S.-Y. Oh). http://dx.doi.org/10.1016/j.biortech.2015.11.021 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

septic systems, atmospheric deposition from nitrogen oxide emissions, and industrial processes. Due to its toxicity to humans (e.g., blue baby syndrome) and the possibility of eutrophication, both the Korean Ministry of Environment and the U.S. Environmental Protection Agency have set a regulation level of 10 mg/L NO3 -N in drinking water. While many techniques have been developed for the treatment of water with exceedingly high concentrations of dissolved NO3 (e.g., ion exchange, reverse osmosis, biological denitrification, chemical reduction), such processes have the disadvantages of being prohibitively expensive, difficult to maintain, and likely to generate concentrated waste (Ahn et al., 2008).

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Zero-valent iron [Fe(0)] has been studied for its ability to reduce NO3 in natural water and wastewater (Westerhoff and James, 2003; Su and Puls, 2004; Yang and Lee, 2005). Fe(0) has also received much attention for the treatment of toxic contaminants because it is abundant, inexpensive, readily available, and has a strong reduction potential (E0h = 0.44 V). Furthermore, the Fe(0) reduction process requires little maintenance, and its corrosion products are relatively innocuous (Oh et al., 2002). Unfortunately, the Fe(0) reduction process for nitrate does not proceed in a straightforward manner. Many researchers have reported that it took several hours or even days to completely remove NO3 by Fe (0) in batch experiments under ambient conditions (Su and Puls, 2004). In other studies, acidic and highly buffered conditions were required to obtain significant NO3 reduction by Fe(0) (Alowitz and Scherer, 2002; Huang and Zhang, 2005). The use of nanoscale Fe(0) (Yang and Lee, 2005) or higher temperatures to overcome the kinetic barrier to reduction (Ahn et al., 2008) has resulted in a significant enhancement of the nitrate reduction rate (half-life range on the order of minutes). While sizable improvements have been achieved in the reduction of nitrate by Fe(0), the reduction products were reported to be mostly NH+4 and thus, requiring additional treatments (e.g., ammonia stripping). Biological denitrification is an attractive nitrate treatment option due to its low cost, high removal rate, and the high specificity of denitrifying bacteria for nitrate (Matéju et al., 1992). Previous studies have shown that Fe(0) can support the microbial reduction of nitrates to nitrogen gas (Till et al., 1998; Shin and Cha, 2008). Shin and Cha (2008) demonstrated that Fe (0) can serve as an electron donor for nitrate reduction by providing H2 to nitrate-respiring bacteria. It has also been suggested that the use of Fe(0) could eliminate the need for a continuous supply of expensive electron donors or hydrogen gas. Hydrogenophilic denitrifiers utilize cathodic hydrogen gas (H2) generated from the anaerobic corrosion of Fe(0) in water to transform nitrate to N2 during batch tests (Shin and Cha, 2008). Hydrogen gas (H2) is one of the most thermodynamically favorable electron donors for nitrate reduction (Till et al., 1998). It is an excellent electron source because of its clean nature and low biomass yield. Moreover, the reaction products of hydrogenophilic denitrification (N2 and water) are innocuous (Smith et al., 1994; Vasiliadou et al., 2006; An et al., 2010). Schaefer et al. (2007) reported the complete removal of 4 mg/L of nitrate-N with Fe(0) and bioculture. The continuous generation of H2 from the Fe(0) corrosion process may overcome the limitations associated with hydrogen-utilizing denitrification, as Fe(0) is a relatively inexpensive H2 source that eliminates the need to handle or store dangerous hydrogen gas. Biochar, a carbon-rich product generated during the pyrolysis of organic materials, has received significant interest in recent years for the production of renewable energy (bio-oil and biogas) and the reduction of CO2 released via carbon sequestration (Lehmann and Joseph, 2009). Biochar has also been increasingly investigated as an adsorbent for organic chemicals and toxic metals in waters and soils (Mizuta et al., 2004; Loganathan et al., 2009; Ahmad et al., 2014; Mohan et al., 2014; Rajapaksha et al., 2014). Due to the presence of functional groups (acidity/basicity) on the biochar surface and p–p electron donor–acceptor (EDA) interactions, toxic contaminants can be sorbed on biochar, thereby decreasing their mobility and bioavailability (Chun et al., 2004). However, enhancements in microbial nitrate reduction in the presence of biochar have not yet been extensively studied. Biochar-amended soil has reportedly been associated with improved absorbency to reduce the leaching of N, P, and organic carbons while increasing the soil surface area (Beck et al., 2011). In addition, higher water retention promotes the probability that microorganisms can uptake nutrients that would otherwise be leached from the soil (Coumaravel et al., 2011). Coelhoso et al. (1992) showed that activated carbon

particles could support wastewater denitrification. Due to their large adsorptive capacity and irregular surface shape, activated carbon particles serve to shelter bacteria from high fluid shear forces. This in turn allows bacteria to form a homogeneous biofilm with a uniform thickness, resulting in more effective denitrification rates. Bock et al. (2015) recently showed that the addition of biochar can significantly enhance nitrate removal in a denitrifying bioreactor. In our previous work, we showed that biochar can act as an electron-transfer mediator to enhance the abiotic reduction of nitro explosives and pesticides by reductants (Oh et al., 2013, 2015). It was proposed that the graphene structure, surface functional groups (e.g., quinone), and possible embedded metals may be responsible for the observed improvement in the reduction of nitro explosives. However, the role of biochar in the biotic transformation of contaminants in the presence of Fe(0) has not yet been examined. In the present study, the microbial denitrification of nitrate in the presence of Fe(0) and biochar was investigated. It was hypothesized that biochar may provide electron-conducting porous structures in order to enhance the anaerobic biotransformation of nitrate by microbes in the presence of Fe(0) by facilitating electron transfer. Through a series of batch experiments, the influence of biochar and Fe(0) addition on microbial denitrification was examined. The effect of the graphene structure was investigated using graphite, a reference black carbon material; the impact of the pyrolysis temperature, biochar dosage, and mixed culture dosage was also examined. Lastly, possible products of denitrification and reduction are discussed. 2. Methods 2.1. Chemicals and materials Potassium nitrate (KNO3, >99%) was purchased from Duksan Pharmaceutical Co. (Kyunggi, Korea). The Fe(0) used in this study was Peerless iron (Peerless Metal Powders and Abrasives, Detroit, MI, USA); the specific surface area of the Peerless iron was 1.67 m2/g, as determined by the Brunauer–Emmett–Teller (BET) method with nitrogen. The mean diameter of the Peerless iron, as evaluated with a size analyzer (Mastersizer 2000, Malvern, UK), was 1.2 mm, while the Fe content was more than 90%. Graphite powder (<20 lm, 99.9%) was obtained from Aldrich (Milwaukee, WI, USA). Biochars were synthesized from rice straw generated in the city of Ulsan. The biochars were pyrolyzed using a laboratory-scale gas flow-controlled tube furnace maintained at 900 °C for 4 h under N2 flowing at a rate of 1000 cc/min. The properties of the graphite and the synthesized biochar (e.g., pH, BET surface area, cation exchange capacity (CEC), point of zero charge (PZC), and elemental composition) are described in a previous report (Oh and Seo, 2014). Mixed cultures were collected from a wastewater treatment facility in the City of Ulsan. The concentration of mixed liquor volatile suspended solids was 10,200 mg/L, while the contents of NO3 -N and NH+4-N in the mixed cultures were 5.6 and 3.3 mg/L, respectively. 2.2. Batch experiments Batch experiments were conducted using 250 mL borosilicate amber bottles. All experimental preparation was carried out in an anaerobic glovebox (JISICO, Seoul, Korea) under ambient N2 conditions. Each amber bottle contained 190 mL of deoxygenated nitrate solution (55 mg/L), 10 mL of mixed culture, 10 g of Fe(0), and 0.5 g of biochar. The bottles were prepared in duplicates to ensure reproducibility and allow for experimental errors to be estimated. MininertÒ valves (VICI Precision Sampling, Baton Rouge, LA, USA)

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2.3. Chemical analysis Nitrate and NH+4 concentrations were quantified by cadmium reduction and salicylate methods (HACH Co., 2007) using a UV–vis spectrophotometer (DR2800, HACH Co., Loveland, CO, USA). Following persulfate digestion, the total nitrogen concentration was also determined with a UV–vis spectrophotometer. The detection limits of NO3 -N, NH+4-N, and total N were 0.6, 1.0, and 2.0 mg/L, respectively (HACH Co., 2007). Solution pH was measured using an Orion 5-star benchtop meter (Thermo Fisher Scientific, Waltham, MA, USA).

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and low-permeability vinyl tape (471, 3M, St. Paul, MN, USA) were employed to seal the bottles and prevent both air intrusion and volatilization losses. The bottles were placed in a horizontal position on a platform shaker rotating at 150 rpm. At selected times, a 1-mL aqueous sample (which possibly included a few fine particles of iron or biochar) was withdrawn using a glass syringe and immediately passed through a 0.20-lm cellulose membrane filter (Millipore, Billerica, MA, USA) to remove particles for soluble nitrate and ammonium concentrations. For every experiment, two sets of control bottles were prepared under identical conditions, one without Fe(0) and the other without biochar. In the reduction control experiments carried out without microbes and biochar, abiotic reduction of the nitrate by Fe(0) was quantified. In the sorption control experiments conducted without microbes and Fe(0), sorption of the nitrate to the biochar was evaluated. Biotic control experiments were also performed under identical conditions without Fe(0) and biochar. In order to ascertain the effect of the graphitic structure, biochar was replaced with graphite, a reference black carbon material. The effect of the pyrolysis temperature was investigated after pyrolyzing biochars at 400, 550, 700, and 900 °C. To determine the effect of the mixed culture dosage, the dosage was increased from 10 to 30 mL under identical conditions.

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3. Results and discussion 3.1. Denitrification of NO3 -N with Fe(0) in the presence of biochar Changes in the concentrations of NO3 -N and NH+4-N in the presence of microbes, Fe(0), and biochar are shown in Fig. 1. During abiotic reduction with Fe(0), only 29% of the nitrate was removed over 14 days. This result is consistent with the slow kinetic values reported in previous studies (Su and Puls, 2004). The concentration of NH+4-N was 1.4 mg/L after 14 days. Because solution pH was not controlled, the pH rapidly increased from 5.8 to 8.9 due to iron corrosion. Since the PZC of Fe(0) is 7.2–7.4, the sorption of nitrate to Fe (0) granules was assumed to be insignificant. In the sorption control experiment, 18% of the nitrate was sorbed to the biochar over 14 days, and the concentration of NH+4-N was less than the detection limit. The presence of biochar rapidly increased the pH to 9.4. Because the PZC of biochar is 11.3 (Oh and Seo, 2015), it appears that the sorption of nitrate to the biochar was favorable. However, the sorptive removal of nitrate from the biochar was limited to 18%, implying that the electrostatic sorption of nitrate to the biochar pyrolyzed at 900 °C is not significant. The co-presence of Fe (0) and biochar resulted in 37% removal of the NO3 over 14 days, indicating an absence of synergistic effects in the removal of nitrate. In contrast, the co-existence of Fe(0) and biochar decreased the removal of nitrate, possibly due to the decrease in nitrate reduction with Fe(0) brought about by the biochar-assisted increase in the solution pH. In the biotic control experiment, the introduction of microbes to the bottle resulted in only 19% removal of the nitrate over 14 days, indicating that biotic transformation of the nitrate was limited under the given conditions. The

Fig. 1. Denitrification of NO3 with Fe(0) and mixed cultures in the presence of biochar pyrolyzed at 900 °C (Fe(0) = 10 g, biochar = 0.5 g, mixed culture = 10 mL (MLVSS = 10,200 mg/L), total solution volume = 200 mL). Data points are the average of duplicate samples, and error bars represent one standard deviation.

introduction of biochar to the microbe-only did not significantly enhance the removal of nitrates. This result is not consistent with previous findings reported by Bock et al. (2015), who showed significant enhancement in nitrate and phosphate removal in a denitrifying bioreactor with biochar addition. A shortage of carbon may account for both the slow biotic transformation of the nitrate and the absence of catalytic effects from the biochar. In contrast to the bioreactor used by Bock et al. (2015), additional carbon source was not added to the batch reactor in the present study. The addition of Fe(0) to the microbes provided a different picture. As previously reported, H2 generated from anaerobic iron corrosion promoted the biotic transformation of nitrate by acting as an electron donor (Till et al., 1998; Shin and Cha, 2008). Over the course of 5 days, 75% of the nitrate was removed by biotic transformation in the presence of Fe(0), while more than 95% of nitrate was removed after 14 days. In addition, the co-presence of Fe(0) and microbes increased the formation of NH+4 as a product. Unlike abiotic removal with Fe(0) and/or biochar, the co-existence of Fe (0) and microbes produced 3.1 mg/L of NH+4-N over 14 days, which is responsible for 26% of the initial N. The introduction of biochar to Fe(0) and microbes further enhanced the microbial transformation of nitrate. Over 3 days, more than 90% of the nitrate was removed, and complete removal of the nitrate was achieved after 7 days. In contrast to the Fe(0)–microbe system, the addition of biochar

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3.2. The effect of pyrolysis temperature In order to determine whether the biochar pyrolysis temperature may affect denitrification kinetics in the microbe–Fe(0)–bio char system, biochars pyrolyzed at 400–900 °C were tested under identical conditions. As shown in Fig. 3, the microbial transformation of nitrate with Fe(0) and biochar pyrolyzed at 550 °C was as rapid as that with 900 °C-pyrolyzed biochar. As the pyrolysis temperature was increased from 400 to 700 °C, the microbial transformation of nitrate did not vary significantly (Fig. 4). Furthermore, no noticeable change in the denitrification rate was observed after comparing the findings to those obtained with graphite. Such results indicate that a pyrolysis temperature between 400 and

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reduced the soluble NH+4 concentrations. After 14 days, only 1.2 mg/L of NH+4-N was formed in the system, indicating that microbial reduction of the nitrate with Fe(0) and biochar was more dominant than abiotic reduction by Fe(0). One possible scenario to explain the lower levels of NH+4 is the sorption of NH+4/NH3 to biochar or iron after the reduction of NO3 to NH+4/NH3 in the Fe(0)–microbe or Fe(0)–microbe–biochar system. However, sorption experiments conducted with NH+4 under identical conditions ruled out this possibility, as the sorptive removal of NH+4 was less than 5% of the initial N. The total N concentration after 14 days in the Fe(0)–microbe–biochar system confirmed that other N-containing compounds did not exist in the aqueous phase. The results suggest that biochar may act as a catalyst to enhance denitrification by microbes in the presence of Fe(0). Torres et al. (2010) previously reported that, during extracellular electron transfer (EET) in microbial fuel cells, anaerobically respiring bacteria may need electron-conducting materials to transfer electrons to solid electron acceptors (e.g., metal oxides, carbon, and metal electrodes). They attributed the high current density to electron transport through a solid conductive matrix. Carbon nanotubes were reported to promote electron transfer from an electrode to microorganisms (Peng et al., 2010). It was also concluded that granular activated carbon facilitates direct interspecies electron transfer between bacteria and methanogens (Liu et al., 2012). Therefore, the beneficial effect of biochar to the Fe(0)–microbe system may be explained by the ability of the electronconducting biochar to enhance microbial transformation of the nitrate. To confirm such behavior, the effect of graphite addition was evaluated under identical conditions. As shown in Fig. 2, the microbial transformation of nitrate in the presence of Fe(0) and graphite was similar to that in the Fe(0)–biochar system. When compared to the control experiments, the addition of graphite to the microbe–Fe(0) system significantly enhanced microbial transformation of the nitrate, with 74% and 93% removal over 3 and 5 days, respectively. This result clearly supports the notion that the improvement in microbial denitrification in the presence of Fe(0)–biochar system is related to the electrical conductivity of black carbon materials. The formation of NH+4 was slightly increased to 4.0 mg/L of NH+4-N after 14 days. Due to increased Fe (0) corrosion in the presence of graphite (initial solution pH = 4.9), abiotic reduction of nitrate by Fe(0) may be more dominant than in biochar systems. It should be noted that the role of functional groups on the surface of the biochar cannot be completely ruled out. It was previously reported that nitrate-reducing bacteria can utilize reduced humic acid as an electron donor (Van Trump et al., 2011). Their experimental results showed that the electron-donating capability of reduced humic acid (e.g., quinone functional groups) was responsible for the microbial transformation of nitrate. Thus, it is still possible that biochars pyrolyzed at 400–700 °C can contain such functional groups, although biochar pyrolyzed at 900 °C does not possess many hydrogen/ oxygen-containing functional groups (Oh and Seo, 2015).

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Time (days) Fig. 2. Denitrification of NO3 with Fe(0) and mixed cultures in the presence of graphite (Fe(0) = 10 g, graphite = 0.5 g, mixed culture = 10 mL (MLVSS = 10,200 mg/ L), total solution volume = 200 mL). Data points are the average of duplicate samples, and error bars represent one standard deviation.

900 °C does not affect the denitrification of nitrate with Fe(0) and biochar. It is well known that the properties of the biochar are strongly affected by the pyrolysis temperature. An elevated pyrolysis temperature resulted in an increase in the electrical conductivity and a decrease in surface functional groups (Oh and Seo, 2015). Therefore, our results suggest that the electrical conductivity of biochar pyrolyzed at 400 °C may be sufficient to accelerate the microbial reduction of nitrate with Fe(0) and biochar, while a further increase of the electrical conductivity may not significantly enhance the microbial transformation. Additional experiments showed that biochar pyrolyzed at 250 °C did not promote microbial denitrification with Fe(0) (Fig. 4). Another possible explanation is that the decreased electrical conductivity of biochar pyrolyzed at low temperatures may be compensated by the formation of surface functional groups, which can act as electron donors. Additional mechanistic studies are needed to further understand the role of biochar pyrolyzed at various temperatures. 3.3. The effect of biochar and mixed culture dosages The effect of the biochar dosage was evaluated in the range of 0.2–1.0 g. An increase in the amount of biochar resulted in higher nitrate removal rate with Fe(0) and microbes. However, considering the sorption of nitrate to biochar, such an enhancement does appear to be significant (Fig. 5). Under the given conditions, it is likely that

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Fig. 5. The effect of the biochar dosage on the denitrification of NO3 with Fe(0) and mixed cultures in the presence of biochar (Fe(0) = 10 g, mixed culture = 10 mL (MLVSS = 10,200 mg/L), total solution volume = 200 mL). Data points are the average of duplicate samples, and error bars represent one standard deviation.

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Fig. 3. Denitrification of NO3 with Fe(0) and mixed cultures in the presence of biochar pyrolyzed at 550 °C (Fe(0) = 10 g, biochar = 0.5 g, mixed culture = 10 mL (MLVSS = 10,200 mg/L), total solution volume = 200 mL). Data points are the average of duplicate samples, and error bars represent one standard deviation.

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Fig. 6. The effect of the mixed culture dosage on the denitrification of NO3 with Fe (0) and mixed cultures in the presence of biochar (Fe(0) = 10 g, biochar = 0.5 g, total solution volume = 200 mL). Data points are the average of duplicate samples, and error bars represent one standard deviation.

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Time (days) Fig. 4. The effect of the biochar pyrolysis temperature on the denitrification of NO3 with Fe(0) and mixed cultures in the presence of biochar (Fe(0) = 10 g, biochar = 0.5 g, mixed culture = 10 mL (MLVSS = 10,200 mg/L), total solution volume = 200 mL). Data points are the average of duplicate samples, and error bars represent one standard deviation.

0.2 g of biochar provides a sufficient number of sites to improve the microbial denitrification of nitrate in the presence of Fe(0). In contrast to the findings described above, the effect of the mixed culture dosage was more pronounced (Fig. 6). In the biotic control experiment conducted with only mixed cultures, an increase in the mixed culture dosage from 10 to 30 mL significantly enhanced the denitrification of nitrate, with complete removal after 5 days. With the addition of Fe(0), a marked improvement in the removal rate was observed. Regardless of the mixed culture dosage, more than 90% of the nitrate was removed within 3 days (Fig. 6). The addition of biochar further increased microbial transformation of the nitrate with Fe(0), as complete nitrate removal was achieved after 3 days. Our results suggest that, under

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the given conditions, the amount of microbes may be the most important factor in enhancing the microbial denitrification of nitrate with Fe(0) and biochar. The importance of mixed culture dosage may also explain that the catalytic role of biochar was not significant in microbial denitrification without Fe(0) (Figs. 1 and 3). Due to the limited amount of microbes (10 mL of mixed culture) without external carbon source, microbial denitrification may not be dominant. The catalytic role of biochar in microbial reduction of nitrate using external carbon source under the given conditions still remains to be determined. 4. Conclusions The addition of Fe(0) to microbes in mixed cultures enhanced the denitrification of nitrate, resulting from Fe(0) corrosion. Further increases in microbial denitrification in the presence of Fe(0) were obtained through the addition of biochar. The enhanced denitrification of nitrate in the microbe–Fe(0)–biochar system primarily due to the presence of biochar. Control experiments conducted with graphite suggested that the electrical conductivity of biochar may be responsible for its catalytic behavior during denitrification. The tasks of optimizing the process and identifying the microbial species responsible for denitrification have yet to be carried out for practical application of the microbe–Fe(0)–biochar system. Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013007767). References Ahmad, M., Rajapaksha, A.U., Lim, J.E., Zhang, M., Bolan, N., Mohan, D., Vithanage, M., Lee, S.S., Ok, Y.S., 2014. Biochar as a sorbent for contaminant management in soil and water: a review. Chemosphere 99, 19–33. Ahn, S.C., Oh, S.Y., Cha, D.K., 2008. Enhanced reduction of nitrate by zero-valent iron at elevated temperatures. J. Hazard. Mater. 156, 17–22. Alowitz, M.J., Scherer, M.M., 2002. Kinetics of nitrate, nitrite and Cr(VI) reduction by iron metal. Environ. Sci. Technol. 36, 299–306. Beck, D.A., Johnson, G.R., Spelok, G.A., 2011. Amending greenroof soil with biochar to affect runoff water quantity and quality. Environ. Pollut. 159, 2111–2118. Bock, E., Smoth, N., Rogers, M., Coleman, B., Reiter, M., Benham, B., Easton, Z.M., 2015. Enhanced nitrate and phosphate removal in a denitrifying bioreactor with biochar. J. Environ. Qual. 44, 605–613. Chun, Y., Sheng, G., Chiu, C.T., Xing, B., 2004. Compositions and sorptive properties of crop residue-derived chars. Environ. Sci. Technol. 38, 4649–4655. Coelhoso, I., Boaventura, R., Rodrigues, A., 1992. Biofilm reactors: an experimental and modeling study of wastewater denitrification in fluidized-bed reactors of activated carbon particles. Biotechnol. Bioeng. 40, 625–633. Coumaravel, K., Santhi, R., Kumar, V.S., Mansour, M.M., 2011. Biochar: a promising soil additive. Agric. Rev. 32, 134–139. Ghafari, S., Hasan, M., Aroua, M.K., 2008. Bio-electrochemical removal of nitrate from water and wastewater – a review. Bioresour. Technol. 99, 3965–3974.

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