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Survival of the Aerobic Denitrifier Pseudomonas Stutzeri

Survival of the Aerobic Denitrifier Pseudomonas Stutzeri

Biosci. Biotechnol. Biochem., 76 (3), 495–500, 2012

Survival of the Aerobic Denitrifier stutzeri TR2 during Co-Culture with Activated Sludge under Denitrifying Conditions

Morio MIYAHARA,1 Sang-Wan KIM,1 Shengmin ZHOU,1;* Shinya FUSHINOBU,1 Takeshi YAMADA,2;** Wakako IKEDA-OHTSUBO,2 Akira WATANABE,3 Keisuke MIYAUCHI,2 y Ginro ENDO,2 Takayoshi WAKAGI,1 and Hirofumi SHOUN1;

1Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 2Faculty of Engineering, Tohoku Gakuin University, Tagajo, Miyagi 985-8537, Japan 3Ebara Engineering Service Co., Ltd., 11-1 Haneda Asahi-cho, Ohta-ku, Tokyo 144-8610, Japan

Received October 17, 2011; Accepted December 5, 2011; Online Publication, March 7, 2012 [doi:10.1271/bbb.110785]

3) The aerobic denitrifier TR2 0.26% per year. Furthermore, N2O is the dominant (strain TR2) has the potential to reduce ozone-depleting substance emitted so far in the 21st emissions during the process. In century.4) this application, it is important to find the best One of the main sources of N2O emissions is competitive survival conditions for strain TR2 in wastewater treatment fields. N2O emission from waste- complex ecosystems. To that end, we examined co- water treatment fields is estimated to be 3.2% of total 5) cultures of strain TR2 with activated sludge via five anthropogenic N2O emission. The biological passage cultures in a medium derived from treated removal process in the fields depends on the activity of piggery wastewater that contained a high concentration nitrifying and denitrifying in the acti- of ammonium. The results are as follows: (i) The vated sludge. Ammonium in the wastewater is first medium supported the proliferation of strain TR2 oxidized aerobically by nitrifying to nitrite (P. stutzeri strains) under denitrifying conditions. (ii) (NO2 ) and nitrate (NO3 ), which are then reduced Nitrite was a better denitrification substrate than nitrate anaerobically by to dinitrogen (N2), for TR2 survival. (iii) Strain TR2 also demonstrated which can be removed from the water. It was long strong survival even under aerobic conditions. This believed that N2O is emitted as a byproduct in both the suggests that strain TR2 is effectively augmented to the nitrifying and the denitrifying process. Recently, we wastewater treatment process, aiding in ammonium- found that N2O emission during an aerobic, nitrifying nitrogen removal and reducing nitrous oxide production process depends on denitrification by ammonia-oxidiz- 6) with a partial nitrification technique in which nitrite ing bacteria (AOB, nitrifiers). This means that the N2O accumulates. emitted during the aerobic process is not a byproduct of nitrification but rather the main product of denitrification Key words: Pseudomonas stutzeri; denitrification; pas- by AOB. In the anaerobic (denitrification) process, the sage culture; respiration; bacterial popula- following conditions have been found to cause N2O tion shift emission: (i) insufficient anaerobiosis, causing inactiva- 7,8) tion of N2O reductase, (ii) the accumulation of high World population quadrupled during the 20th century concentrations of nitrite,9) and (iii) a lack of electron and is still increasing. This rapid increase in population donors (a low COD/N ratio).10) The accumulation of occurred in parallel with an increase in the amount of nitrite is artificially realized in the wastewater treatment chemically synthesized ammonia used in nitrogen process due to the use of a partial nitrification fertilizer, which in turn contributed to an accelerating technique,11,12) in which a high concentration of ammo- rise in food production.1,2) The worldwide spread of nium, derived from piggery wastes, for example,13) is tremendous amounts of fixed nitrogen has caused many efficiently removed. However, heavy emission of N2O environmental problems, among which the global is a drawback of the technique that must be improved 9) emission of nitrous oxide (N2O) gas is the most serious. upon. N2O is a the global warming potential of It was long believed that denitrification is an which relative to (CO2) is 298-fold over anaerobic process, and, as such, repressed under aerobic a 100-year time horizon, and its concentration in the conditions,14) but several aerobic denitrifiers have been atmosphere continues to increase, by approximately identified.15,16) However, there is no study concerning

y To whom correspondence should be addressed. Tel/Fax: +81-3-5841-5148; E-mail: [email protected] * Present address: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan ** Present address: Department of Environmental and Life Sciences, Toyohashi University of Technology, Tempaku-cho, Toyohashi 441-8580, Japan Abbreviations: AOB, ammonia-oxidizing bacteria; LB, Luria-Bertani; MLM, mixed liquor medium; MSBR, membrane sequencing batch bioreactor; DLMF, digestion liquid after methane fermentation of piggery wastewater; BOD, biological demand; SSCP, single strand conformation polymorphism; OD, optical density 496 M. MIYAHARA et al. aerobic denitrifiers that has examined whether they emit Culture conditions. Seed culture and preculture of strain TR2 were N O under aerobic, denitrifying conditions. We use the conducted in LB medium under aerobic (non-denitrifying) conditions, 2 18) term ‘‘aerobic denitrifier’’ to designate denitrifiers that as previously described. The activated sludge was grown in MSBR (liquid volume, 30 L; hydraulic retention time, 30 d; sludge retention perform complete denitrification to the formation of N2 time, 45 d; nitrogen loading, approximately 300 mg-N L1 day1), and even under aerobic conditions without emitting N2O. the operating cycle alternated between aerobic and denitrification We have isolated Pseudomonas stutzeri TR2 (strain (anoxic) phases. A portion of the sludge was sampled just before the TR2) as a candidate aerobic denitrifier and characterized end of the denitrifying phase for use in this study. The main passage it.17) Strain TR2 has a strong aerobic denitrification cultures containing activated sludge were conducted as follows: Nine mL of MSBR-activated sludge (MLSS 10 g L1) supplemented with capability accompanied by a low level of N2O emission even in the presence of high concentrations of toxic strain TR2 (activated sludge:strain TR2 ratio 100:1 wet weight) was inoculated into 9 mL of MLM medium18) in an L-shaped test tube nitrite. We further found that the low N2O emission of (Monod tube, 27 mL in volume). For aerobic cultures, the MLM was strain TR2 is dependent on its preference for N2Oas supplemented with sodium acetate (10 mM), and the test tube was together with constitutive expression plugged with an air-permeable foam plug. For denitrifying cultures, the 15 15 of the nosZ (N2O reductase) even under non- MLM was supplemented with Na NO2 (20 mM)orNa NO3 (20 mM) denitrifying, aerobic conditions.18) Hence we suggested and sodium acetate (10 mM). After purging of the headspace of the tube with argon gas and replacement of the argon portion (1%) with O2 gas that strain TR2 should be useful for reducing N2O emissions when used to augment wastewater treatment by the use of a gas-tight syringe (Hamilton, Reno, NV), the tube was 18) sealed with a double butyl rubber stopper. For both the aerobic and the processes that tend to emit large amounts of N2O. denitrifying culture, the tubes were shaken at 120 rpm on a reciprocal Nevertheless, there has been little work on applying shaker at 30 C for 36 h. After the first culture, a portion (9 mL) of the such a bacterial strain to wastewater treatment fields to culture broth was transferred to a fresh medium and cultured again in reduce N2O emissions (bioaugmentation). To the best the same manner (the second culture). Thus the culture passage was of our knowledge, only nitrite oxidizing bacteria have repeated 5 times. The gas phase analysis and measurement of the 18) successfully been applied to a composting process to optimal density of the culture have been previously described. 19) reduce N2O emissions. Thus it is a pioneering Quantitative real-time PCR analysis. A primer set specific for the challenge to apply strain TR2 as a bioaugmentation 0 agent during the wastewater treatment process. For the 16S rRNA gene of strain TR2, Pseu 136f (5 -TGATAGTGGGGGA- CAACG-30) and Pseu 598r (50-GGGGCTTTCACATTCAAC-30), was successful application of strain TR2 to wastewater designed. To check primer specificity, the genomic DNA of various treatments, it is important to keep the bacterium denitrifiers used in our previous study (P. stutzeri ZoBell ATCC dominant in mixed activated sludge cultures. In this 14405, JCM 2412, Ralstonia pickettii K50, study, we examined co-cultures of strain TR2 with and Paracoccus denitrificans JCM 20620) as well as the total DNA of activated sludge and monitored the population of the the MSBR-grown sludge were used as templates for real-time PCR, strain during five culture passages under aerobic and it was confirmed that any non-specific bands were not amplified. A universal primer set comprising EUB (eubacteria) 338f (50-ACTCC- (nitrifying) and anaerobic (denitrifying) conditions. TACGGGAGGCAGC-30)20) and 907r (50-CCGTCAATTCMTTTRA- GTT-30)21) was used to amplify the total bacterial 16S rRNA gene.22) Materials and Methods The cell suspensions from the passage culture (9 mL) and pure culture of the strain TR2 (0.5 mL) were harvested and placed into a 1.5-mL Bacterial strain and media. Pseudomonas stutzeri strain TR2 was tube before storage at 20 C. The genomic DNA was extracted and isolated in a previous study.17) Luria-Bertani (LB) medium was used purified by phenol-chloroform treatment and precipitation as for seed culture and preculture. Mixed liquor medium (MLM), derived previously described,23,24) and the concentration of DNA was adjusted from piggery wastewater,18) was used to passage cultures to mimic a to 5 ng mL1. The DNA samples used in the generation of the standard wastewater treatment field. MLM was prepared by mixing membrane curve for real-time PCR analysis were prepared as follows: The sequencing batch bioreactor (MSBR)-treated water (pH 8.3) and genomic DNA of strain TR2 was amplified using KAPA2G Fast DNA digestion liquid after methane fermentation of piggery wastewater polymerase (Kapa Biosystems, Woburn, MA) with a 16S rRNA gene (DLMF) (pH 8.3) at a ratio of 59:1, followed by supplementation with primer set comprising 27f (50-AGAGTTTGATCMTGGCTCAG-30)21) a trace element solution. The biological oxygen demand (BOD)/total and 1492r (50-GGTTACCTTGTTACGACTT-30).25) The PCR product nitrogen ratio of the DLMF was 0.34. The compositions of the DLMF was purified by extracting the bands obtained by agarose gel and MSBR-treated water are shown in Table 1. The MLM contained electrophoresis, and a dilution series (50 mL of each dilution) was 2 8 1 approximately 5 mM ammonium and low levels of organic matter, constructed (10 to 10 copies mL ) using an Easy Dilution Kit including organic nitrogen compounds. This medium was adjusted to (Takara, Ohtsu, Japan). Real-time PCR was performed using SYBR pH 6.8 and was sterilized by filtration (pore size, 0.2 mm). The details Premix Ex Taq (Takara) and a LightCycler machine (Roche of the preparation of the MLM medium and the MSBR-activated Diagnostics, Tokyo) under the following conditions: initial denatura- sludge, which was grown in MLM were described previously.18) tion at 95 C for 10 s, followed by 35 cycles of 95 C for 5 s, 66 C (TR2 specific primer) or 50 C (bacterial universal primer) for 20 s, and Table 1. Concentrations of Wastewater Solute Contents (mg L1)in 72 C for 15 s. the Basal Solutions Used in the Preparation of MLM PCR-SSCP (single strand conformation polymorphism) analysis. Solute contents DLMF MSBR-treated watera PCR-SSCP analysis was conducted as described by Hori et al., with 26,27) 0 BOD 3,010 —b minor modifications. A primer set comprising B342If (5 - 0 28) 0 Soluble-BOD 1,220 —b CTACGGGIGGCIGCAGT-3 ) and U806Ir (5 -GGACTACCIGGG- 0 29) 0 Organic nitrogen 896 —b TITCTAA-3 ) was used, and the 5 -end of the reverse primer was Ammonium-nitrogen 3,980 60.2 phosphorylated. Bacterial 16S rRNA were amplified from the Nitrate-nitrogen N.D.c N.D.c sample after each culture passage using KAPA2G Fast DNA Polymer- c c ase. PCR was conducted by the following protocol: initial denaturation Nitrite-nitrogen N.D. N.D. Total nitrogen 8,850 160 at 95 C for 3 min, followed by 35 cycles of 95 C for 3 min (denaturation), 50 C for 30 s (annealing), 72 C for 30 s (extension), aMeasured at the end of the denitrifying phase. and a final extension for 30 s at 72 C. Amplification of the targeted bNot measured. PCR product was confirmed by agarose gel electrophoresis. To obtain c Not detected. single-strand DNA, the PCR product was purified using a LaboPass Co-Culture of P. stutzeri Strain TR2 and Activated Sludge 497 PCR purification Kit (Hokkaido System Science, Sapporo) and digested with 10 U lambda exonuclease (New England BioLabs, A D Beverly, MA) at 37 C for 1 h in a final volume of 50 mL. The product was further processed by phenol-chloroform extraction and ethanol precipitation, and then dissolved in 25 mLof10mM Tris–HCl (pH 8.0). The amount of DNA remaining was confirmed by applying part of the sample to agarose gel electrophoresis. Twelve mL of the treated DNA was mixed with the same volume of denaturing loading buffer (95% v/v formamide, 10 mM NaOH, 0.025% w/v bromophenol blue, and 0.025% w/v xylene cyanol). The mixture was incubated at 95 C for 2 min and then cooled on ice. The polyacrylamide gel for electro- phoresis was prepared using gel matrix (MDE; FMC Bioproducts, B E Rockland, ME) diluted 0.675-fold. After the addition of 16.2 mL tetramethylethylenediamine and 162 mL of 10% (w/v) ammonium persulfate, the gel was cast for 1 h on a forming unit 16 cm in length and width and 1 mm in thickness. The loading buffer was 0:5 TBE (45 mM Tris–HCl, 45 mM boric acid, 1 mM, and EDTA, pH 8.0), and electrophoresis was performed at 450 V at 20 C for 15 h by the DCode System (Bio-Rad Laboratories, Hercules, CA). The bands were visualized using an EzStain Silver staining kit (Atto Corp., Tokyo).

Purification of SSCP bands, cloning, and sequencing. The selected C F SSCP bands were exercised with a clean razor blade. The DNA sample was extracted and purified using a QIAEX Gel Extraction Kit (Qiagen, Hilden, Germany) and dissolved in 10 mM Tris–HCl (pH 8.0). Each of the extracted DNA samples was examined again by PCR-SSCP to confirm that the isolated sample constituted a single band. The DNA sample was then used as template for PCR amplification, and amplification of the target gene was confirmed by agarose gel electrophoresis. The excised gel was purified using a LaboPass Gel Extraction and Purification Kit (Hokkaido System Science). The purified DNA was ligated into pT7blue-2 vector (Merck, Fig. 1. during the Five Passage Cultures. Darmstadt, Germany) using a Ligation High Kit (Toyobo, Osaka, Cultures of MSBR-grown activated sludge supplemented with 1% Japan), and then transformed into DH5 competent strain TR2 (TR2-plus; A, B, and C, open symbols) and cultures of cells. DNA sequencing was performed with pT7blue-2 forward activated sludge only (TR2-minus; D, E, and F, closed symbols) in (50-ACTGCAGACGCGTTACGTATCGG-30) and M13 reverse (50- MLM containing 10 mM sodium acetate. Aerobic cultures (A and D) ACGTCGTGACTGGGAAAAC-30) sequence primers and the CEQ and denitrifying cultures (argon-purged headspace with 1% O2) 15 2000XL DNA Analysis System (Beckman Coulter, Fullerton, CA). An supplemented with 20 mM Na NO2 (B and E) or with 20 mM 15 analysis of the sequence data and sequence similarity searches were Na NO3 (C and F). Symbols denoting the culture passage numbers performed using the BLAST program of the National Center for are as follows: circle, first; triangle, second; diamond, third; square, Biotechnology Information (Bethesda, MD). fourth; and asterisk, fifth.

Results and Discussion stationary phase, changed under aerobic conditions (Fig. 1A), suggesting that the microbial flora also Passage cultures of strain TR2 in competition with changed. activated sludge At an early stage of TR2-minus culture growth, cell The survival ability of strain TR2 in a complex growth was very slow (Fig. 1D–F), and micro-aggrega- ecosystem was examined by co-culturing it with tions (floc) were observed (data not shown). The slow activated sludge. Consecutive culture passages were increase in the optical density (OD) was due to initiated by inoculating simultaneously strain TR2 and flocculation of the cells. At the following stages, the excess activated sludge (1:100 wet weight) and this was population of free-living cells increased, which yielded a repeated 5 times (see ‘‘Materials and Methods’’), and rapid increase in OD. Under the aerobic (Fig. 1D) and growth during each passage under varying conditions nitrite-denitrifying (Fig. 1E) conditions, rapid increases was monitored (Fig. 1). A culture without the addition in the number of free-living cells was soon attained from of strain TR2 (TR2-minus culture) was also performed the second passage culture, whereas a long time were as control (Fig. 1D–F). The control culture exhibited required for the nitrate-denitrifying culture to attain a poor growth at an early stage of passaging, with a long rapid growth rate (Fig. 1F). lag time for growth (Fig. 1D–F). By contrast, rapid growth was attained from the initial stage of culture Denitrification by passage cultures when strain TR2 was added (TR2-plus culture) Denitrification during the culture passages under (Fig. 1A–C). These results suggest that strain TR2 or denitrifying conditions was monitored using 15N-labeled 15 other bacteria grew rapidly under these conditions, and substrates. Time-dependent N2-emissions (Fig. 2) are consistent with our previous observation that strain coincided with cellular growth (Fig. 1), indicating that TR2 grows rapidly in MLM medium under both aerobic growth depended on denitrification. Denitrification by and denitrifying conditions.18) In the TR2-plus culture, the TR2-plus cultures was active from the first culture the growth profile was steady throughout the passages (Fig. 2A and B), whereas the TR2-minus cultures under denitrifying conditions (Fig. 1B and C), suggest- showed a longer lag at the early stages (Fig. 2C and 15 ing that the microbial flora were soon stabilized. By D). No culture reached complete denitrification ( N2, contrast, the profile of , in particular at the 5 mmol L1), because the electron donor was limited. 498 M. MIYAHARA et al.

AC AB C

BD Fig. 3. Survival of Strain TR2 during the Five Passage Cultures. The TR2/EUB ratio (strain TR2 per total bacteria) was determined by measuring the copy numbers of their 16S rRNA genes by real-time PCR analysis. At least three replicate measure- ments were performed, and standard deviations are indicated as error bars. The sample before to the first passage culture (A, the sample before the first passage culture), TR2-plus (B), and TR2-minus (C) cultures. Symbols: gray columns, zero-time passage culture (S, sludge only; S+T, TR2-plus; ND, not detectable); white column, aerobic passage cultures; black column, nitrite-denitrifying passage Fig. 2. Denitrification of the Five Passage Cultures. cultures. 15 N2 produced by the denitrifying cultures shown in Fig. 1B, C, E, and F. TR2-plus cultures (open symbols) with nitrite (A) and nitrate (B), and TR2-minus cultures (closed symbols) with nitrite (C) ditions. It is intriguing that some TR2-sibling species and nitrate (D). Symbols denoting the passage culture numbers are appeared even in the TR2-minus culture and that the follows: circle, first; triangle, second; diamond, third; square, fourth; and asterisk, fifth. bacterial species survived throughout the culture pas- sages under denitrifying conditions (Fig. 3C). The species, however, did not show explosive proliferation, The recovery (yield) of N2 from nitrite (85% Fig. 2A unlike strain TR2 (Fig. 3B), suggesting that they are and C) was approximately 2-fold higher than the N2 related to but not the same as strain TR2. yield from nitrate (45% Fig. 2B and D), which agrees 30) with the theoretical values. N2O emissions were Bacterial population analysis of passage cultures by detected only in the first passage culture under nitrite- PCR-SSCP denitrifying conditions. The total amount of N2O The bacterial population after each passage culture emitted from the TR2-plus culture (0.12 mmol L1) was was identified by PCR-SSCP analysis (Fig. 4). The significantly lower than the amount emitted from the dominant bacteria consisted of several species belonging TR2-minus culture (0.85 mmol L1). Therefore, both the to the genera Acinetobacter (bands 1 and 2) and tendency of the nitrite-denitrification process to emit Pseudomonas (bands 3 and 4). The major bands (bands N2O readily along with the N2O emission-reducing 1–4) in a given lane contained identical nucleotide effects of strain TR2 augmentation, which we observed sequences (data not shown), suggesting that bands of the previously,18) were confirmed in this study. same mobility in a given lane originated from the same bacterial strains grown in that passage culture. The Confirmation of the survival of strain TR2 during the nucleotide sequence of the DNA extracted from a given culture passages SSCP band indicated highest identity to the species, Before cultivation, the strain TR2-specific 16S rRNA as follows: band 1, 99% identity (396/399 bp) to gene was detected only in the TR2-plus culture A. seohaensis strain BSD 30 (GenBank accession no. (Fig. 3A), and the ratio of the TR2-specific gene to the DQ518593); band 2, 98% identity (363/367 bp) to total amount of the 16Sr RNA gene of eubacteria (TR2/ A. seohaensis strain E-12 (FJ392126); band 3, 99% EUB) (0.46%) agreed well within the experimental error identity (410/413 bp) to P. stutzeri strain NF-11 for the inoculum size (1%). On the other hand, the strain (FJ527558); and TR2 marker (lane T) and band 4, TR2-specific DNA was not detected in the TR2-minus 100% (390/390 bp) identity to P. stutzeri strain TR2. As culture, suggesting that the population of TR2-sibling a control, PCR-SSCP analysis of the activated sludge species was very small in the MSBR-grown activated before passage culture was also done. Many very thin sludge. The TR2/EUB ratio in the TR2-plus culture bands appeared in the analysis (data not shown), but increased enormously during the first culture under both none of them became dominant, indicating that the conditions (Fig. 3B), although the preculture of strain major band (bands 1–4) species became dominant TR2 was preformed in another medium (LB), in contrast during the passage cultures. to the preculture of the activated sludge in MLM. The Strain TR2 (band 4) proliferated as a dominant ratio decreased significantly after the fourth passage species from the first TR2-plus passage cultures under culture under aerobic conditions (Fig. 3B), while the both aerobic and denitrifying conditions (Fig. 4A and ratio did not decrease throughout the five passage B), and it remained dominant throughout the culture cultures under nitrite-denitrifying conditions. A result passaging process under denitrifying conditions similar to that under nitrite-denitrifying conditions was (Fig. 4B). On the other hand, the strain TR2 band obtained for the culture under nitrate-denitrifying con- disappeared in the final (fifth) passage culture under ditions (data not shown). These results indicate that aerobic conditions (Fig. 4A). These results are consis- strain TR2 can survive only under denitrifying con- tent with the results in Figs. 1 and 3, and confirm that Co-Culture of P. stutzeri Strain TR2 and Activated Sludge 499

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Fig. 4. Population Shift of Passage Cultures as Analyzed by PCR-SSCP. Aerobic TR2-plus (A), nitrite-denitrifying TR2-plus (B), aerobic TR2-minus (C), and nitrite-denitrifying TR2-minus (D) cultures. Lane numbers correspond to numbers of culture passages. One sample from each passage culture was analyzed. Symbols: T, marker of strain TR2; bands 1 and 2, genus Acinetobacter; bands 3 and 4, genus Pseudomonas. The details of bacterial identification are described in the text. Black arrows denote sequenced bands. strain TR2 survived throughout the culture passages other hand, P. stutzeri was not isolated from an enrich- under denitrifying conditions, but it did not survive ment culture under aerobic denitrifying conditions in a under aerobic conditions. In the aerobic TR2-plus medium that was rich in organic nitrogen (8.6 g L1 of culture, band 2 (Acinetobacter) became dominant by peptone).36) The culture medium used in this study the final passage culture, suggesting that strain TR2 was (MLM) was derived from digestion liquid after methane expelled by competition with this bacterium. Bands 1 fermentation of piggery wastewater (DLMF) (see and 2 appeared as dominant species only under aerobic ‘‘Materials and Methods’’), which is also poor in organic conditions (Fig. 4A and C), suggesting that these nitrogen (below 100 mg L1), because organic matter is Acinetobacter species are obligate aerobes.31) The exhausted by methane fermentation. Thus this substrate results also suggest that facultative anaerobes, such as appears to be suitable for the survival of P. stutzeri.Itis strain TR2, cannot overcome obligate aerobes during noteworthy that P. stutzeri TR2 or TR2-sibling strain competition for survival under continuous aerobic became dominant also in the TR2-minus culture more conditions. It is of interest, however, that although band rapidly under nitrite-denitrifying conditions than under 1 was the most dominant band observed in the TR2- nitrate-denitrifying conditions (Figs. 1E and F, 2C and minus aerobic culture (Fig. 4C), it disappeared in the D, and 4), suggesting that the high concentration final TR2-plus aerobic culture (Fig. 4A). These results (20 mM) of toxic nitrite occasioned selective pressure might reflect severe competition between the band 1 that was advantageous to the survival of the P. stutzeri strain and strain TR2, together with a strong survival TR2 and/or its relative strains. A few bacterial species ability for strain TR2 even under non-denitrifying, that are tolerant to nitrite rapidly became dominant aerobic conditions. under nitrite-denitrifying conditions (Fig. 1E) due to Both bacterial species that survived and were dom- weak competition. On the other hand, the preference of inant during competition under nitrite-denitrifying con- other many bacterial species for nitrate over nitrite as ditions in the TR2-minus passage culture (bands 3 and 4, substrate resulted in severe competition for survival Fig. 4D) were identified as P. stutzeri based on PCR- under nitrate-denitrifying conditions (Fig. 1F). Hence SSCP analysis. Furthermore, one of the P. stutzeri a longer time was required for the dominant species to strains (the band 4 strain) appeared to be very closely be selected. related to strain TR2, since the nucleotide sequence of The results of the present study indicated several its PCR-SSCP fragment was completely identical to that characteristics of strain TR2 and related P. stutzeri of the corresponding portion of the 16S rRNA gene of species. (i) Denitrifying conditions in a medium con- strain TR2. This means that a P. stutzeri strain TR2- taining ammonium ions and low levels of organic sibling species can become dominant in the passage nitrogen, such as MLM, are advantageous for them to culture of activated sludge even without inoculating become dominant. (ii) Nitrite is better than nitrate as a strain TR2. However, some differences were observed denitrification substrate for P. stutzeri survival, because between the phenotypes of the TR2-plus and TR2-minus weaker competition is expected (Figs. 1E and F, 2C and passage cultures under nitrite- and nitrate-denitrifying D). (iii) In MLM medium, strain TR2 exhibits high conditions (Figs. 1–4). It must be clarified by future survival ability even under continuous aerobic condi- study whether the P. stutzeri strain that appeared under tions, although it is finally defeated (Fig. 4A). It can be the TR2-minus nitrite-denitrifying condition (band 4; concluded that the application of strain TR2 to reduce 18) Fig. 4D) is entirely the same strain as TR2 or rather a N2O emissions is most effective when the strain is newly grown sibling strain. used to augment sewage disposal of wastewater that P. stutzeri strains are recognized as active denitrify- contains low COD and high levels of ammonium, such ing heterotrophic bacteria.32) It has been found that as DLMF. The strain is best used in conjunction with a P. stutzeri proliferates under denitrifying conditions in a partial nitrification technique, such as a denitrification- medium that is poor in organic nitrogen and supple- nitrification liquid circulating reactor9) or a nitrite- mented with nitrate or nitrite (electron acceptors) and accumulating intermittently aerated sequence batch organic acid or hydrogen (electron donors).33–35) On the reactor.12) Under such wastewater treatment conditions, 500 M. MIYAHARA et al. the microorganisms are exposed to severe conditions 12) Zhang M, Lawlor PG, Wu G, Lynch B, and Zhan X, Bioprocess Biosyst. Eng., 34, 1049–1056 (2011). that normally N2O emission, viz., high concen- trations of nitrite and ammonium, a low COD/N ratio, 13) Bernet N, Delgenes N, and Moletta R, Environ. Technol., 17, 293–300 (1996). and intermittent aeration (recurring aerobic and anoxic 14) Betlach MR, Antonie Van Leeuwenhoek, 48, 585–607 (1982). conditions). These conditions are, however, advanta- 15) Kim JK, Park KJ, Cho KS, Nam SW, Park TJ, and Bajpai R, geous for the survival of strain TR2, and the strain Bioresour. Technol., 96, 1897–1906 (2005). should be able to display an ability to reduce N2O 16) Kong QX, Wang XW, Jin M, Shen ZQ, and Li JW, FEMS emissions under such conditions. Microbiol. Lett., 260, 150–155 (2006). 17) Takaya N, Catalan-Sakairi MA, Sakaguchi Y, Kato I, Zhou Z, Acknowledgments and Shoun H, Appl. Environ. 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