Bioaugmentation of a wastewater bioreactor system with the nitrous oxide-reducing denitrifier Pseudomonas stutzeri strain TR2

Journal of Bioscience and Bioengineering VOL. 115 No. 1, 37e42, 2013 www.elsevier.com/locate/jbiosc

Bioaugmentation of a wastewater bioreactor system with the nitrous oxide-reducing denitrifier Pseudomonas stutzeri strain TR2 Wakako Ikeda-Ohtsubo,1 Morio Miyahara,2 Sang-Wan Kim,2 Takeshi Yamada,1, x Masaki Matsuoka,2 Akira Watanabe,3 Shinya Fushinobu,2 Takayoshi Wakagi,2 Hirofumi Shoun,2 Keisuke Miyauchi,1 and Ginro Endo1, * Faculty of Engineering, Tohoku Gakuin University, 1-13-1 Chuo, Tagajo, Miyagi 985-8537, Japan,1 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,2 and Ebara Engineering Service Co., Ltd, 11-1 Haneda Asahi-cho, Ohta-ku, Tokyo 144-8610, Japan3 Received 20 June 2012; accepted 20 August 2012 Available online 19 September 2012

In bioaugmentation technology, survival of inoculant in the treatment system is prerequisite but remains to be a crucial hurdle. In this study, we bioaugmented the denitrification tank of a piggery wastewater treatment system with the denitrifying bacterium Pseudomonas stutzeri strain TR2 in two pilot-scale experiments, with the aim of reducing nitrous oxide (N2O), a gas of environmental concern. In the laboratory, strain TR2 grew well and survived with high concentrations of nitrite (5e10 mM) at a wide range of temperatures (28e40 C). In the first augmentation of the pilotscale experiment, strain TR2 inoculated into the denitrification tank with conditions (30 C, w0.1 mM nitrite) survived only 2e5 days. In contrast, in the second augmentation with conditions determined to be favorable for the growth of the bacterium in the laboratory (40e45 C, 2e5 mM nitrite), strain TR2 survived longer than 32 days. During the time when the presence of strain TR2 was confirmed by quantitative real-time PCR, N2O emission was maintained at a low level even under nitrite-accumulating conditions in the denitrification and nitrification tanks, which provided indirect evidence that strain TR2 can reduce N2O in the pilot-scale system. Our results documented the effective application of growth conditions favorable for strain TR2 determined in the laboratory to maintain growth and performance of this strain in the pilot-scale reactor system and the decrease of N2O emission as the consequence. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Bioaugmentation; Wastewater treatment; Denitrification; Inoculant survival; Quantitative real-time PCR]

Nitrogen removal from wastewater is an important process to avoid eutrophication caused by excess nitrogen flow into the environment from sewage and fertilizers. Conventional nitrogen removal process involves microbial nitrification and denitrification processes, in which aerobic nitrifying bacteria oxidize ammonia to nitrate (NO3  ) or nitrite (NO2  ), which in turn are reduced to gaseous nitrogen, mostly N2O and N2, by denitrifying microorganisms under anoxic conditions. In conventional piggery waste management, anaerobic digestion of organic substances in the wastewater is applied after solids and liquids are separated, leading to acquisition of the reusable biogas methane (1). Such anaerobic digestion of piggery waste is widely used and has a number of advantages, such as less sludge residue and higher energy recovery (2); however, the digested effluent contains high amounts of ammonia (1e10 g total N L1), which must be removed to meet the strict discharge regulations. Various activated sludge systems have been designed for optimizing nitrogen removal from wastewater (3), but these systems may become a significant source of N2O because a small amount of * Corresponding author. Tel.: þ81 22 368 7493; fax: þ81 22 368 7070. E-mail address: [email protected] (G. Endo). x Present address: Department of Ecological Engineering, Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku-cho, Toyohashi 441-8580, Japan.

dissolved oxygen unavoidably remains in the anoxic reactor and may inhibit the reduction of N2O to N2 by denitrifying bacteria (4). Furthermore, the microoxic conditions also allow nitrifying bacteria, such as Nitrosomonas sp., to perform nitrifier denitrification, which is believed to be a main source of N2O in some environments (5,6). N2O is a potent greenhouse gas and considered to be the most important substance involved in ozone depletion (7). The source of N2O emission is mainly attributed to biological origins in labile environments, e.g., agricultural soils; therefore, controlling N2O emission is more challenging than controlling CO2 emission, which can be reduced by various methods, such as fuel saving and forestation. Since some denitrifying bacteria and most nitrifying bacteria as well as denitrifying fungi emit N2O as the end product of denitrification (8e11), it is important to select and utilize denitrifying bacteria that reduce but do not emit N2O in bioaugmentation of the wastewater treatment system. Recently, we have documented that the aerobic denitrifying bacterium Pseudomonas stutzeri strain TR2 is able to grow by denitrification and to reduce N2O even under oxic conditions (12). Under microoxic conditions (3% O2), P. stutzeri strain TR2 preferentially utilizes electron acceptors in the order N2O > nitrite > nitrate (12). This prominent N2O-reducing ability of P. stutzeri strain TR2 led us to

1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.08.015

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consider using the bacterium for bioaugmentation of a denitrifying reactor to reduce N2O emission. Bioaugmentation is a cost-effective environmental technology that involves inoculation of cultivated microorganisms into a target environment, such as a wastewater treatment system, to enhance overall microbiological activity. It is often used for degrading toxic recalcitrant compounds such as organochlorines to harmless compounds (13,14), but also has been applied for improving sludge performance (15), enhancing the tolerance of the microbial community against various stresses (16), or protecting an indigenous microbial population from a shock load of a toxic recalcitrant substrate (17). In bioreactor systems, many unsuccessful cases of bioaugmentation have been reported, and the recognized cause of failure is low survival of the augmented strain in the indigenous microbial community (18,19). To achieve an ideal outcome from bioaugmentation, therefore, it is critical to adjust conditions of the target reactor so that the inoculant remains viable and functionally active. We previously demonstrated that the survival in passage cultures and concomitant N2O reduction of P. stutzeri strain TR2 significantly improved under nitrite-denitrifying conditions as compared to nitrate-denitrifying conditions (20). In the present study, we bioaugmented a newlydeveloped pilot-scale piggery wastewater treatment system with P. stutzeri strain TR2 under two different conditions, one unaltered and the other altered to the advantage of for strain TR2. Our results show that the use of growth conditions favorable for the augmented strain TR2 determined in our laboratory experiments increased the effectiveness of the bioaugmentation.

MATERIALS AND METHODS Growth of P. stutzeri TR2 under various conditions P. stutzeri strain TR2 was previously isolated from rice paddy soil (21). A 100 mL pre-culture of strain TR2 (OD600 ¼ 0.9e1.1) grown overnight in LB medium was inoculated into Hungate tubes with a headspace of 2.5% O2 in Ar and containing 10 mL 1/5 LB medium (2 g Bacto tryptone, 1 g yeast extract, and 5 g sodium chloride per liter) or DM medium (21) supplemented with NaNO2 or NaNO3 as an electron acceptor. The tubes were incubated (i) at 30 C with different concentrations (0, 0.2, 1, 5, 10, 20 mM) of NaNO2, or (ii) at different temperatures (15e42 C) with 10 mM NaNO2 or NaNO3. The optical density (OD) at 600 nm was measured periodically using a spectrophotometer (NOVASPEC II, Pharmacia, LKB Biochrom, UK) and a biophotometric recorder (TN-2612, Advantec Toyo, Japan). The OD values of triplicate culture tubes for each condition were plotted against the incubation time to obtain a growth curve for each growth condition. The specific growth rate was calculated from the slope of each growth curve. N2O in the headspace of test tubes of 24-h cultures was measured with a Porapak Q column by gas chromatography (TCD, GC-14A, Shimadzu, Japan). Pilot reactor setup The pilot-scale reactor system (Fig. 1) was installed at a piggery farm. Liquid piggery manure partially anaerobically digested via a methane fermentation tank flowed directly into the system. The influent flowed first into an anoxic denitrification tank (4 m3), and the effluent from the tank flowed into an oxic nitrification tank (4 m3). A part of the effluent from the oxic tank flowed into a heating tank (0.5 m3). The remaining effluent flowed into a membrane filtration tank (0.5 m3) and returned to the denitrification tank. The filtered effluent from the membrane filtration tank and the excess sludge were separately discharged from the plant. The heating tank was introduced in this system to facilitate fermentation and solubilization of organic substances available for denitrifiers and to eliminate heat-susceptible nitrite-oxidizing bacteria to allow denitrification via nitrite, suitable for survival of strain TR2 (21,22). The heating tank was constantly maintained at 40e45 C using the excess heat produced from the methane fermentation tank and heat from ammonia oxidation. The denitrification tank and nitrification tank maintained a pH of 7.8e8.1 and 7.6e8.3, respectively, which are both in the range of optimum growth pH for strain TR2 (data not shown). A dissolved oxygen (DO) concentration in the denitrification tank was <0.1 mg L1, and an oxidation/reduction potential (ORP) was kept between 457 and 31 mV. The soluble biochemical oxygen demand (S-BOD; w15,000 mg L1) and the ammonia concentration (2500e4900 mg L1) of the influent of the denitrification tank were significantly higher than the S-BOD (2.6e20 mg L1) and ammonia concentration (0.30e76.5 mg L1) of the effluent from the heating tank, which indicated that the soluble organic substances were consumed in the denitrification/nitrification tanks. In the nitrification tank, NH4eN conversion rate was 1.65 kg/m3/day in average.

J. BIOSCI. BIOENG.,

FIG. 1. Schematic diagram of the experimental setup of a pilot-scale piggery wastewater treatment system: 1, influent (methane-digestion liquor); 2, influent (liquid manure); 3, anoxic denitrification tank; 4, oxic nitrification tank; 5, heating tank; 6, membrane filtration tank; 7, excess sludge; 8, water discharge; P, water pump; AP, air pump. Bioaugmentation A large-scale pre-culture (150 L) of P. stutzeri TR2 was prepared in LB medium (1% tryptone, 0.5% yeast extract, 5% NaCl) with 0.25% antifoam (Wako, Osaka, Japan) using culture bags (Fujimori Kogyo Co. Ltd., Tokyo, Japan) bubbled with an air pump. A 150 L aliquot of the pre-culture (OD 2.0e2.5) was harvested by centrifugation, and the cells were resuspended in 20 L of water treated by a membrane sequence batch reactor (20) and stored at 4 C. Two bioaugmentation experiments were carried out, one 34 days and one 127 days after the wastewater treatment system began to operate stably. The cell suspension of strain TR2 accounted for 0.22% and 0.25% of the total volume of activated sludge in the first and second bioaugmentation, respectively, and was directly added to the denitrification tank. Analytical methods The physicochemical properties of the anoxic denitrification tank were sequentially measured as follows. DO and temperature were monitored using a dissolved oxygen analyzer (DO402G, Yokogawa Electric Co., Tokyo, Japan). The ORP was monitored using an OR100 Analyzer (Yokogawa Electric Co.); the pH was measured using a PH100 pH meter (Yokogawa Electric Co.). N2O emission was measured with a multi-gas analyzer (VA-3000, Horiba, Japan). Volatile total solid and volatile suspended solid were determined according to standard methods (23). Biochemical oxygen demand (BOD) and S-BOD were determined using a dissolved oxygen meter (B-103Z, Iijima Electronics Corp., Japan). The concentration of nitrogen in the form of nitrate (NO3eN) and in the form of nitrite (NO2eN) was measured by a spectrometric continuous flow analyzer (QuAAtro2-HR, BL Tec K. K., Japan). Quantitative real-time PCR Activated sludge was sampled from the denitrifying reactor in the following time periods: first augmentation at 5 min, 2 h, and 1, 2, 6, and 9 days after augmentation with strain TR2; second augmentation immediately before and after the augmentation, and then at 3 h, 1e8 days, and 11, 13, 18, 25, and 32 days after augmentation with strain TR2. Each sludge sample was pelleted in a centrifuge tube, and total DNA was prepared from each sample using FastDNA SPIN kit (BIO 101, Carlsbad, CA, USA) according to the manufacturer’s instructions, and subsequently resuspended in 100 mL of deionized water. The quality and quantity of the DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA). A 10 ng sample of sludge DNA was mixed with SYBR Premix Ex Taq (Perfect Real Time, Takara Corp., Shiga, Japan) and the following primer pairs (10 pmol each): for 16S rRNA genes of strain TR2, Pseu 136f and Pseu 598r (20); for 16S rRNA genes of Bacteria, EUB 338f (24) and EUB 907r (25). Standard DNA for calibration was prepared from a serial dilution of 16S rRNA gene fragments amplified from strain TR2 using the primer set 27f (26) and 1492r (27). 16S rRNA genes were quantified by real-time PCR using a thermocycler and continuous fluorescence monitoring (LightCycler; Roche Diagnostics, Switzerland) under the following conditions: an initial denaturation step (10 s, 95 C), followed by 45 cycles of repeated denaturation (10 s at 95 C), annealing (10 s, 50 C for Bacteria or 66 C for strain TR2), and polymerization (15 s at 72 C). Each protocol included a negative control (DNase-free water), a seven-point external standard, and a standard for calibration. The size of each real-time PCR product was checked on a 1% agarose gel with a 100-bp standard ladder (Takara Corp.).

RESULTS Effect of nitrite concentration on the growth of P. stutzeri strain TR2 As our pilot-scale denitrification reactor was operated under nitrite-accumulating conditions, which is favorable for

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Effect of temperature on the growth of P. stutzeri strain TR2 The pilot-scale reactor system used in this bioaugmentation study employed a heating tank to elevate the temperature of the denitrification tank mixed liquor up to 40 C. Therefore, we determined the influence of temperature on the growth of P. stutzeri strain TR2 under denitrifying conditions at the nitrate or nitrite concentration of 10 mM. The optimum growth rate of strain TR2 was obtained at 30 C when either nitrate or nitrite was used as a denitrification substrate (Fig. 3). No growth was observed above 45 C. At the higher range of temperatures (37e43 C), the growth rate decreased when strain TR2 was grown in 1/5 LB medium supplemented with nitrate. In contrast, the growth of strain TR2 was not as much affected when nitrite was provided, which indicated that strain TR2 was more resistant to high temperature stress during growth with nitrite. In the lower range of temperatures (15e25 C), the growth rate of strain TR2 was slightly higher in cultures with nitrate than in those with nitrite. In this experiment, a small amount of N2O (w100 ppm) in the headspace was detected only in cultures containing nitrate (data not shown). Changes in temperature, nitrite concentration, and N2O emission in the pilot reactor after bioaugmentation with P. stutzeri strain TR2 We bioaugmented our pilot-scale reactor system with P. stutzeri strain TR2 in two experiments. The culture of strain TR2 was added to the denitrification tank 34 and 127 days after the wastewater treatment system began to operate stably. Fig. 4 shows the changes in the profiles of temperature, the amount

0.2

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0.12 20 mM

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1 mM 0.2 mM

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25

FIG. 2. Effect of the concentration of nitrite as an electron acceptor on the growth of P. stutzeri TR2 in DM medium. The optical density (OD) at 600 nm was measured twice in triplicate, and mean values were plotted against the time after inoculation with strain TR2. Error bars are not shown because the standard deviation did not exceed 0.002 among the corresponding values except a few reads, which fluctuation was largely attributed to the initial instability of the inoculated cultures in each experiment.

0.25

0.2 Growth rate (1/h)

P. stutzeri strain TR2, we investigated how the nitrite concentration affects the growth of this strain. In our batch culture experiments, the highest growth rate was observed at a nitrite concentration of 5 mM and 10 mM (Fig. 2). The optimum growth yield, i.e., the maximum OD, was obtained at a nitrite concentration of 10 mM, but initiation of growth was delayed compared to growth with 5 mM nitrite. A nitrite concentration of 20 mM inhibited the growth of the strain TR2, i.e., both the growth rate and growth yield were lower. Strain TR2 did not grow well at nitrate concentrations <1 mM, but the growth rate was not affected at 1 mM nitrate. Under all conditions tested, N2O in the headspace was under the detection limit (details not shown).

39

0.15

0.1

0.05

0 0

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20 30 Temperature (°C)

40

50

FIG. 3. Effect of temperature on the growth rate of P. stutzeri strain TR2 in 1/5 LB medium with 10 mM nitrate (open circle) or nitrite (filled triangle) as an electron acceptor. The growth rate (m) was calculated from the measured OD values (N) during the exponential growth phase using the equation: ln (Nt)  ln (No) ¼ m (t  to). All values are the average of triplicate measurements, and error bars represent the standard deviation.

of N2O emission in the denitrification tank, NO2eN concentrations in both the nitrification and denitrification tanks, and NO3eN concentration in the denitrification tank. On the day of the first augmentation (day 34), the temperature of the heating tank was temporarily lowered to bring the denitrification tank to near the optimum growth temperature of strain TR2 (32e34 C). This, contrary to our intentions, resulted in the reduction of the nitrite concentration to 0.1 mM (<4.6 mg L1) in the denitrification tank at the point of bioaugmentation. Although the emission of N2O stopped immediately after strain TR2 was introduced into the denitrification tank, N2O became detectable again when the nitrite concentration in the nitrification and denitrification tanks increased as the heat treatment tank restored 30 days after the bioaugmentation. In the second augmentation (day 127), the operational setting was changed toward conditions favorable for the growth of the strain TR2 based on the results of laboratory growth experiments (see above). Ten days prior to the second bioaugmentation until the 3rd day after, we added sodium nitrite solution (5 kg NaNO2/day) into the denitrification tank by a continuous flow pump to reach a nitrite concentration of 2e5 mM (100e250 mg L1). The temperature of the heating tank was maintained at 40e45 C before bioaugmentation, and the temperature of the denitrification tank ranged between 37 C and 40 C. In the second augmentation, nitrite concentration in the nitrification and denitrification tanks reached its peak (227 and 199 mg L1, respectively) on the next day and there was a short-term increase of N2O emission in the denitrification tank on days 130 and 131, i.e., 3 and 4 days after the augmentation (Fig. 4). However, after day 132, N2O emission was maintained at a lower level for more than 30 days even when the nitrification and denitrification tanks accumulated nitrite (Fig. 4). This was in clear contrast to the first augmentation and the operation before the two experiments, in which increases of nitrite concentration in the nitrification and denitrification tanks always coincided with significant N2O emission (Fig. 4). Survival of P. stutzeri strain TR2 in the denitrification tank To investigate the survival of P. stutzeri strain TR2 in the denitrification tank, we quantified 16S rRNA genes of Bacteria and of strain TR2 in the sludge DNA by real-time quantitative PCR. In the first augmentation (day 34), the copy number of strain TR2 16S rRNA genes rapidly decreased on day 36 (2 days after the augmentation) to <1% of the copy number calculated from the volume of strain TR2 added to the reactor (6.48  105 per 1 mg

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500

50

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40 35

Inorganic N-compounds (mg/L)

300

30 25 20

200

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N2O (ppm v/v)

45

15 100

10 5

0

0

50

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0

Operational time (days) FIG. 4. Profiles of temperature (solid line), N2O concentration (ppm v/v) in the sampled gas (filled circle), NO2eN concentration (mg/L) in the denitrification tank (open triangle) and in the nitrification tank (open square), and NO3eN concentration (mg/L) in the denitrification tank (cross) during the operation of the pilot plant. The dotted line indicates a 2-point moving average of the N2O concentration in the denitrification tank. The open arrows indicate the periods when the presence of P. stutzeri strain TR2 in the denitrification tank was able to be detected by real-time quantitative PCR (see Fig. 5).

sludge DNA) and then was under the detection limit (<103 per 1 mg sludge DNA) on day 40 (6 days after the augmentation), whereas there was no change in the total copy number of bacterial 16S rRNA genes (w1010 per 1 mg sludge DNA). The pilot plant was operated at solids retention times (SRT) ranging from 18 to 21 days and hydraulic retention times (HRT) from 8 to 9 days, therefore the disappearance of strain TR2 in the first augmentation was not attributed to a washout. In contrast, in the second bioaugmentation (day 127), the copy number of 16S rRNA genes of strain TR2 decreased at a lower rate and even occasionally increased (Fig. 5). They were continuously detected at 3.85  107e6.56  104 copies

DISCUSSION

1.0E+11 Copy number of 16S rRNA genes/ 1 µg sludge DNA

per 1 mg sludge DNA between days 127 and 159 (0 and 32 days after the bioaugmentation) (Fig. 5). This indicated that with conditions favorable for growth, the survival of strain TR2 increased approximately eightfold. Similar quantities of TR2 16S rRNA genes were also detected in the activated sludge samples from the nitrification tank for the same period of time (data not shown), indicating that strain TR2 survived also in the nitrification tank and may have provided back to the denitrification tank in a viable condition. When N2O emission increased on days 180e190 (Fig. 4), 16S rRNA genes of strain TR2 were under the detection limit in the sludge samples (data not shown).

1.0E+09

1.0E+07

1.0E+05

1.0E+03

Elapsed time after bioaugmentation

FIG. 5. Changes in the copy number of 16S rRNA genes of total Bacteria (open bars) and those of P. stutzeri TR2 (filled bars) per 1 mg sludge DNA in the denitrification tank after the second bioaugmentation. Note that 1 mg DNA accounted for 5.3e15.6 mg activated sludge (wet weight), i.e., 0.53e1.56 MLSS (mg L1). Error bars indicate the standard deviation.

In this study, the optimum growth conditions determined in laboratory experiments were effectively applied to maintain the growth and performance of P. stutzeri strain TR2 bioaugmented into our pilot-scale wastewater treatment system. We have previously reported that P. stutzeri strain TR2 is able to outcompete other denitrifying bacteria, such as Paracoccus denitrificans, and survives well in a successive culture under nitrite-denitrifying conditions (12,20). In addition to these findings, our laboratory investigation presented here revealed that strain TR2 grows optimally when the medium contains 5e10 mM nitrite, while it grows poorly at concentrations <1 mM or >20 mM (Fig. 2). The addition of sodium nitrite before the second bioaugmentation increased the nitrite concentration in the nitrification and denitrification tanks and this has dramatically improved the survival of strain TR2 (Fig. 5), underlining that the nitrite concentration is a critical factor for the growth and survival of strain TR2 also on larger scale processes. The growth experiments also revealed that strain TR2 tolerates a higher range of temperatures under nitrite-denitrifying conditions (Fig. 3). The prominent ability of strain TR2 to use nitrite as an electron acceptor even at a high temperature probably enabled this strain to out-compete other microorganisms including many bacteria and protozoa,

VOL. 115, 2013 which are susceptible to nitrite toxicity and high temperatures. Preventing the growth of protozoa is especially of great importance since the rapid disappearance of strain TR2 after the first augmentation probably resulted from grazing by protozoa because we consistently observed protozoa in our pilot system (data not shown), and grazing has often been claimed to be the main cause of failure in bioaugmentation (18). In this study, N2O emission was maintained at a low level during the time period when 16S rRNA genes of strain TR2 were detectable, which suggested that strain TR2 in the reactor repressed N2O formation or removed N2O. An increasing nitrite concentration in both denitrification and nitrification stages has been recognized as a cause of N2O emission from wastewater treatment systems (4). Nevertheless, in this study nitrite accumulation in the nitrification and denitrification tanks did not result in N2O emission in the second augmentation, whereas increases of nitrite concentration before the second augmentation were always followed by N2O emission. The cause of N2O emission and reduction that occurred during 3e5 days after the second bioaugmentation is not clear, but we assume it may reflect a period of time required for strain TR2 to adapt the environment in the denitrification tank. Considering that no 16S rRNA genes of strain TR2 were detected in the sludge samples 60 days after the second bioaugmentation, constant addition of both nitrite and a culture of strain TR2 into the denitrification tank should be necessary at least on a bimonthly basis to maintain the strain TR2 population and its effect in the pilot-scale system. To our knowledge, this study is the first report of bioaugmentation aiming at reducing N2O using a pilot-scale reactor system. A few studies of bioaugmentation have used denitrifying bacterial strains and bench-scale reactors (e.g., Ref. 28), but these studies have focused only on improving the efficiency of denitrification and did not fully investigate the growth conditions of the inoculant. In contrast, our study is based on a through investigation on the growth characteristics of P. stutzeri strain TR2 (12, 20; and this study), and we developed our own pilot-scale wastewater treatment system, which enabled us to determine the condition, which is favorable to strain TR2 and applicable both for batch culture and bioaugmentation. Nevertheless, to validate the effect of bioaugmentation on the reduction of N2O emission quantitatively, the target system should be improved so that a certain amount of N2O is constantly emitted because sporadic and unpredictable emission of N2O is usually observed in controlled wastewater treatment systems (4). It is also important to elucidate the mechanism of N2O emission from each target system by studying its unique microbial community structure responsible for denitrification. An alternative approach to nitrogen removal from livestock wastewater with low N2O emission is SHARON/Anammox process (29), which combines single reactor system for high activity ammonia removal over nitrite (SHARON) for partial oxidation of ammonium to nitrite, with anaerobic ammonia oxidation (Anammox) for anaerobic oxidization of ammonium to nitrogen gas with nitrite as the electron acceptor. However, this type of process requires strict control of DO, C/N ratio (30), ammonia/ nitrite ratio (31), otherwise it can be easily out-competed by heterotrophic denitrification producing N2O. Therefore, further development is necessary for any wastewater treatment systems to achieve nitrogen removal without N2O emission. As has been argued elsewhere, a body of failed bioaugmentation studies in the past largely lacks fundamental investigation of the microbial strains and the target habitat (32). Although it is a ‘rule of thumb’ that small-scale experiences are not directly applicable to a largescale environment, this study validated the importance of basic laboratory-scale experiments to achieve positive results in a scaled-up system.

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ACKNOWLEDGMENTS This study was supported by Research and Development Program for New Bio-industry Initiatives and Grant-in-Aids for Scientific Research from the Japan Society for the Promotion of Science (to HS, No. 20248009; to GE, No. 21360258). We thank Kentaro Orita at The University of Tokyo as well as Sanae Shiratori and Kunihito Saito at Tohoku Gakuin University for their assistance in laboratory experiments.

References 1. Andreadakis, A. D.: Anaerobic digestion of piggery wastes, Water Sci. Technol., 25, 9e16 (1992). 2. Mata-Alvarez, J., Mace, S., and Llabres, P.: Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives, Bioresour. Technol., 74, 3e16 (2000). 3. Seviour, R. K., Lindrea, C., Griffiths, P. C., and Blackall, L. L.: The activated sludge process, pp. 448e475, in: Seviour, R. J. and Blackall, L. L. (Eds.), The microbiology of activated sludge. Kluwer Academic Publications, Dordrecht, The Netherlands (1999). 4. Kampschreur, M. J., Temmink, H., Kleerebezem, R., Jetten, M. S. M., and Van Loosdrecht, M. C. M.: Nitrous oxide emission during wastewater treatment, Water Res., 43, 4093e4103 (2009). 5. Itokawa, H., Hanaki, K., and Matsuo, T.: Nitrous oxide production in highloading biological nitrogen removal process under low COD/N ratio condition, Water Sci. Technol., 35, 657e664 (2001). 6. Shaw, L. J., Nicol, G. W., Smith, Z., Fear, J., Prosser, J. I., and Baggs, E. M.: Nitrosospira spp. can produce nitrous oxide via a nitrifier denitrification pathway, Environ. Microbiol., 8, 214e222 (2006). 7. Ravishankara, A. R., Daniel, J. S., and Portmann, R. W.: Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century, Science, 326, 123e125 (2009). 8. Greenberg, E. P. and Becker, G. E.: Nitrous oxide as end product of denitrification by strains of fluorescent pseudomonads, Can. J. Microbiol., 23, 903e907 (1977). 9. Kool, D. M., Wrage, N., Zechmeister-Boltenstern, S., Pfeffer, M., Brus, D., Oenema, O., and Van Groenigen, J. W.: Nitrifier denitrification can be a source of N2O from soil: a revised approach to the dual-isotope labelling method, Eur. J. Soil Sci., 61, 759e772 (2010). 10. Kim, S. W., Miyahara, M., Fushinobu, S., Wakagi, T., and Shoun, H.: Nitrous oxide emission from nitrifying activated sludge dependent on denitrification by ammonia-oxidizing bacteria, Bioresour. Technol., 101, 3958e3963 (2010). 11. Shoun, H., Fushinobu, S., Jiang, L., Kim, S.-W., and Wakagi, T.: Fungal denitrification and nitric oxide reductase cytochrome P450nor, Phil. Trans. R. Soc. B., 367, 1186e1194 (2012). 12. Miyahara, M., Kim, S.-W., Fushinobu, S., Takaki, K., Yamada, T., Watanabe, A., Miyauchi, K., Endo, G., Wakagi, T., and Shoun, H.: Aerobic denitrification by Pseudomonas stutzeri TR2 has the potential to reduce nitrous oxide emission from wastewater treatment plants, Appl. Environ. Microbiol., 76, 4619e4625 (2010). 13. Boon, N., Goris, J., De Vos, P., Verstraete, W., and Top, E. M.: Bioaugmentation of activated sludge by an indigenous 3-chloroaniline degrading Comamonas testosteroni strain, I2gfp, Appl. Environ. Microbiol., 66, 2906e2913 (2000). 14. Major, D. W., McMaster, M. L., Cox, E. E., Edwards, E. A., Dworatzek, S. M., Hendrickson, E. R., Starr, M. G., Payne, J. A., and Buonamici, L. W.: Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene, Environ. Sci. Technol., 36, 5106e5116 (2002). 15. Jiao, Y., Zhao, Q., Jin, W., Hao, X., and You, S.: Bioaugmentation of a biological contact oxidation ditch with indigenous nitrifying bacteria for in situ remediation of nitrogen-rich stream water, Bioresour. Technol., 102, 990e995 (2011). 16. Head, M. A. and Oleszkiewicz, J. A.: Bioaugmentation for nitrification at cold temperatures, Water Res., 38, 523e530 (2004). 17. Boon, N., Top, E. M., Verstraete, W., and Siciliano, S. D.: Bioaugmentation as a tool to protect the structure and function of an activated-sludge microbial community against a 3-chloroaniline shock load, Appl. Environ. Microbiol., 69, 1511e1520 (2003). 18. Bouchez, T., Patureau, D., Dabert, P., Juretschko, S., Doré, J., Delgenès, P., Moletta, R., and Wagner, M.: Ecological study of a bioaugmentation failure, Environ. Microbiol., 2, 179e190 (2000). 19. Yu, L., Peng, D., and Ren, Y.: Protozoan predation on nitrification performance and microbial community during bioaugmentation, Bioresour. Technol., 102, 10855e10860 (2011). 20. Miyahara, M., Kim, S.-W., Zhou, S., Fushinobu, S., Yamada, T., IkedaOhtsubo, W., Watanabe, A., Miyauchi, K., Endo, G., Wakagi, T., and

42

21.

22.

23.

24.

25.

26.

IKEDA-OHTSUBO ET AL. Shoun, H.: Survival of the aerobic denitrifier Pseudomonas stutzeri strain TR2 during co-culture with activated sludge under denitrifying conditions, Biosci. Biotechnol. Biochem., 76, 495e500 (2012). Takaya, N., Catalan-Sakairi, M. A., Sakaguchi, Y., Kato, I., Zhou, Z., and Shoun, H.: Aerobic denitrifying bacteria that produce low levels of nitrous oxide, Appl. Environ. Microbiol., 69, 3152e3157 (2003). Bougard, D., Bernet, N., Chèneby, D., and Delgenès, J.-P.: Nitrification of a high-strength wastewater in an inverse turbulent bed reactor: effect of temperature on nitrite accumulation, Process Biochem., 41, 106e113 (2006). Eaton, A. D., Clesceri, L. S., and Greenburg, A. E.: Standard methods for the examination of water and wastewater, pp. 4e23. American Public Health Association (APHA), Washington, DC (1995). Amann, R. I., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R., and Stahl, D. A.: Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations, Appl. Environ. Microbiol., 56, 1919e1925 (1990). Lane, D. J.: 16S/23S rRNA sequencing, pp. 115e175, in: Stackebrandt, E. and Goodfellow, M. (Eds.), Nucleic acid techniques in bacterial systematics. Wiley, Chichester (1991). Edwards, U., Rogall, T., Blöcker, H., Emde, M., and Böttger, E. C.: Isolation and direct complete nucleotide determination of entire genes. Characterization of

J. BIOSCI. BIOENG.,

27.

28.

29.

30.

31.

32.

a gene coding for 16S ribosomal RNA, Nucleic Acids Res., 17, 7843e7853 (1989). Weisburg, W. G., Barns, S. M., Pelletier, D. A., and Lane, D. J.: 16S ribosomal DNA amplification for phylogenetic study, J. Bacteriol., 173, 697e703 (1991). Park, K. Y., Kim, S. J., Jung, J. Y., and Lee, S. H.: Reduction of N2O emission from biological nitrogen removal processes by Alcaligenes faecalis augmentation, J. Ind. Eng. Chem., 13, 508e511 (2007). van Dongen, U., Jetten, M. S. M., and van Loosdrecht, M. C. M.: The SHARONÒeAnammoxÒ process for treatment of ammonium rich wastewater, Water Sci. Technol., 44, 153e160 (2001). van Hulle, S., Vandeweyer, B. D., Meesschaert, P. A., Vanrolleghem, P., and Dumoulin, A.: Engineering aspects and practical application of autotrophic nitrogen removal from nitrogen rich streams, Chem. Eng. J., 162, 1e20 (2010). Muangthong-on, T. and Wantawin, C.: Evaluation of N2O production from anaerobic ammonium oxidation (Anammox) at different influent ammonia to nitrite ratios, Energy Procedia, 9, 7e14 (2011). Thompson, I. P., van der Gast, C. J., Ciric, L., and Singer, A. C.: Bioaugmentation for bioremediation: the challenge of strain selection, Environ. Microbiol., 7, 909e915 (2005).