Soil Biology & Biochemistry 42 (2010) 1721e1727

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Soil Biology & Biochemistry

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Different responses of denitrication rates and denitrifying bacterial communities to hydrologic pulsing in created wetlands
Keunyea Song a, b, c, Seung-Hoon Lee a, William J. Mitsch b, Hojeong Kang a, *
a

School of Civil and Environmental Engineering, Yonsei University, Shinchon 134, Seodaemun-gu, Seoul 120-749, Republic of Korea Wilma H. Schiermeier Olentangy River Wetland Research Park, The Ohio State University, 352 W. Dodridge Street, Columbus, OH 43202, USA c Department of Environmental Engineering, Ewha Womans University, Deahyundong 11-1, Seodaemun-gu, Seoul 120-750, Republic of Korea
b

a r t i c l e i n f o
Article history: Received 8 February 2010 Received in revised form 10 June 2010 Accepted 10 June 2010 Available online 2 July 2010 Keywords: Hydrologic pulsing Created wetlands Denitrication Denitrifying bacterial community structure Olentangy River Wetland Research Park

a b s t r a c t
Hydrologic pulsing, including water level drawdown and subsequent ooding, may have a considerable impact on both biogeochemical processes and microbial communities in wetlands. Since denitrifying bacteria play a key role in water quality improvement in wetlands, changes in their activities and communities with hydrologic pulsing are an important issue. We investigated the responses of in situ denitrication rates, denitrifying bacterial community structure and their quantities using nitrite reductase (nir) S gene under different hydrological pulsing conditions in created wetlands in central Ohio USA. Average denitrication rates, measured from 4 different sampling locations, were 302, 133, 71 and 271 mg N2OeN m2 h1 during inundated, saturated, drying and reooding periods, respectively. In particular, the denitrication rates in shallow water level marsh areas (SM) followed by deepwater level marsh areas (DM) showed more sensitivity and magnitude of changes to hydrologic pulsing events than did non-vegetated deepwater areas. This may have been due to the high aerobic decomposition during the drying period and nutrient ushing in shallower marsh areas after the reooding event. In contrast, the community structure and diversity of denitriers based on terminal-restricted fragment length polymorphism (T-RFLP) analysis showed no signicant change due to hydrologic pulsing. Instead, the presence and absence of vegetation altered denitrifying bacterial community structure. The nirS gene copy number remained relatively constant with only minor increases during water level drawdown followed by a signicant decrease when a sudden reooding event occurred. These results indicate that environmental disturbances, such as hydrologic pulsing, have a major impact on the denitrication process, but less impact on the community structures of the denitrifying bacteria. In addition, there was no relationship among the denitrication rate, the community structure, and the quantity of denitriers, suggesting that changes in denitrication rates during hydrologic pulsing events were not caused by the changes in microbial community structure but more by physicochemical factors, such as substrate availability and hydrology. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Hydrologic changes, such as summer and autumn drought followed by ooding, are now expected to be more severe in rivers and their adjacent riparian ecosystems due to changes in the precipitation patterns as well as riparian buffer zone (IPCC, 2001; Mitsch and Jrgensen, 2004). In particular, since hydrology is considered to be the single most inuential factor determining wetland characteristics (Mitsch and Gosselink, 2007), altered

* Corresponding author. Tel.: 82 2 2123 5803; fax: 82 2 364 5300. E-mail address: hj_kang@yonsei.ac.kr (H. Kang). 0038-0717/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.06.007

hydrologic regime could be responsible for drastic changes in structures and functions of wetlands. Several studies have reported the effect of hydrologic changes on wetlands in terms of their structure and ecological function. In general, enhanced mineralization rates and substantial nutrient leaching are well known consequences of successive drying and ooding (Baldwin and Mitchell, 2000; Turner and Haygarth, 2001; Venterink et al., 2002). These altered process rates under hydrologic changes could be associated with the responses of microbial communities (Schimel and Gulledge, 1998). It has been also suggested that changes in microbial communities and their biomass caused changes in biogeochemical processes such as methane emissions and soil respiration in wetlands (Freeman et al., 2002; Davidson et al., 2004; Knorr et al., 2008). Likewise, Kim et al.

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(2008) reported that different types of wetlands may exhibit diverse responses to drought in terms of denitriers and methanogens. However, the question of whether the microbial community structure affects processes at the ecosystem scale has provoked much debate with conicting results (e.g. Cavigelli and Robertson, 2000; Loreau et al., 2001; Rich et al., 2003; Dandie et al., 2008; Ma et al., 2008). The effects of pulsing hydrology on water quality, denitrication, nitrous oxide and methane emissions, sedimentation, and aquatic productivity were investigated in a comprehensive pulsing study at the created wetlands in 2004e2005 used in this current study sitedThe Olentangy River Wetland Research Park in central Ohio USA (Mitsch et al., 2005, 2008; Hernandez and Mitsch, 2006, 2007; Altor and Mitsch, 2006, 2008; Nahlik and Mitsch, 2008; Tuttle et al., 2008). Our current study on denitrication and microbial communities in the same wetlands is a follow-up to those investigations of the importance of river pulsing on wetland ecosystem function. One of the important processes occurring in wetlands is denitrication, which transfers nitrate to gas type nitrogen (Paul and Clark, 1996; Zumft, 1997). The denitrication process has been studied as a means of natural water quality improvement and a source/sink of nitrous oxide (e.g. Lashof and Ahuja, 1990; Hernandez and Mitsch, 2006, 2007), and denitrifying bacterial communities have been investigated in natural environments (e.g. Braker et al., 2001; Castro-Gonzlez et al., 2005: Cao et al., 2008). Wallenstein et al. (2006) suggested that denitrifying bacterial communities in soils are controlled by environmental conditions such as temperature and moisture conditions as proximal control factors and by environmental disturbances as distal factors. Although several researchers have already studied denitrifying bacterial community structure in wetlands, how those communities respond to environmental disturbances such as the drying and reooding of hydrologic pulses, and their relationship with denitrication rates in wetlands remains poorly understood. Denitrication is mediated by denitrifying bacteria communities under anaerobic conditions (Zumft, 1997; Philippot and Hallin, 2005), hence, it is expected that hydrologic pulsing could affect both denitrifying bacterial community structure and the denitrication process, subsequently. In addition, hydrologic pulsing can act as a physiological stress and disturbance for microorganisms (Baldwin and Mitchell, 2000; Schimel et al., 2007; Gordon et al., 2008) possibly resulting in a decrease in quantity of denitrifying bacteria. Therefore, we investigated how the denitrication process and denitrifying bacterial community structure and quantity responded to hydrologic pulsing events in created wetlands. We also tested the relationships among denitrifying bacterial community structure, their quantity, and denitrication rates to better understand the role of microbial community structure at the ecosystem scale. Since denitrication is catalyzed by nitrite reductase (nir), which is widespread among taxonomically diverse microorganisms (Philippot, 2002), we targeted nitrite reductase (nir) S gene as a marker indicating the presence of denitrifying bacteria. We used the terminal-restricted fragment length polymorphism (T-RFLP) technique and real-time PCR (RT-PCR) to analyze the denitrifying bacterial community structure and denitrifying bacterial quantity, respectively. 2. Materials and methods 2.1. Site description and experimental designs This study was conducted at the Wilma H. Schiermeier Olentangy River Wetland Research Park (ORWRP) placed at The Ohio State University in Columbus, USA (Fig. 1). Two 1-ha wetland areas

were created in 1993e1994 and have had river water pumped through them continuously since then (Mitsch et al., 1998, 2005, 2008). The sampling sites in both wetlands were selected based on both a longitudinal gradient from inow to outow and a transverse gradient from deepwater in the middle to shallow water on the edges (Fig. 1). The sampling sites included the inow and outow deepwater areas along the longitudinal gradient and the deepwater (DM) and shallow water (SM) marsh sites along the transverse gradient. DM and SM sites were selected near inow and outow areas. The two wetlands were used as replicates and each sampling site had 2 pseudo-replicates. To investigate hydrologic pulses, we sampled denitrication rates and microbial communities during 4 different hydrologic phases of a drying and rewetting cycle in each of the two wetlands: inundated, saturated, drying and reooding. The inundated phase in July 2008 occurred before water level drawdown commenced and was considered a control treatment. In early August 2008, surface water level decreased naturally due to low precipitation for 2 weeks; this was considered to be the saturated phase. Then, pumped water inow from the river to the wetlands was purposefully discontinued for 7 days from August 23 to 29, 2008, and both wetland dried-up; this is the drying phase. After the week of drying, water was pumped again into the wetlands for the reooding phase. It took only a day to ood the wetland areas to their initial water depths. The water depths for each phase are presented in Table 1. 2.2. Water and soil sampling and measurements Water temperature, pH, and DO were measured in situ using a YSI-meter. Duplicated soil and water samples from each site were collected and stored at 4  C before subsequent analysis. Soil samples for molecular analysis were preserved at 20  C. Extracted water from soil samples with double distilled water were ltered through 0.45 mm lter and used to determine dissolved organic

Fig. 1. Two 1-ha experimental wetland basins used in this study at the Olentangy River Wetland Research Park at the Ohio State University, Columbus, USA. Open circle, triangle, rectangle and diamond represent inow deepwater (IN), deepwater marsh (DM), shallow water marsh (SM) and outow deepwater (OUT), respectively. IN and OUT sampling site had four replicates while DM and SM had eight replicates in two created wetlands.

K. Song et al. / Soil Biology & Biochemistry 42 (2010) 1721e1727 Table 1 Water level (mean SE) in each hydrologic phase in the simulated pulse in the created experimental wetlands at the Olentangy River Wetland Research Park.a Surface water level (cm) Inundated Saturated Drying Reooding IN 6.7 1.3 3.0 5.6 0.0 0.5 0.7 0.0 DM 5.6 0.8 8.0 5.8 0.0 0.8 0.4 0.1 SM 0.6 0.5 7.0 3.1 0.4 0.5 0.4 0.5 OUT 7.4 5.5 0.3 6.1 0.4 2.1 0.1 0.1

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a IN, DM, SM and OUT represent different sampling sites: inow area, deepwater marsh, shallow water marsh and outow area of the two wetlands, respectively.

The 20 ml reaction mixture contained template DNA, 2 SYBR green Supermix (Bio-Rad) and primer pair (each nal conc. 12.5 mM) nirS832F (TAC CAC CCC GAG CCG CGC GT) e nirS3R (GCC GCC GTC RTG VAG GAA). The thermal cycling conditions of the RT-PCR included denaturation at 95  C for 2 min, 40 cycles of amplication at 95  C for 25 s, 65  C for 30 s and 72  C for 25 s. The PCR was performed using an I-Cycler Version 3.0 a (Bio-Rad) RT-PCR assay was performed two times for each duplicated sample. 2.5. Statistical analysis

carbon (DOC) concentrations (mg g soil1) using TOC-meter (TOCV, Shimadzu). Specic UV absorbance (SUVA) was calculated using the same ltered water samples by UV absorbance at 254 nm (USEPA, 2005). Extractable nitrate contents (mg g soil1) in the sediment were analyzed by the colorimetric method (Anderson and Ingram, 1989). In situ denitrication rates in the presence of hydrologic pulsing events were measured using the acetylene blocking technique (Tiedje, 1982; Tiedje et al., 1989). PVC chambers, 4 cm in diameter and 80 cm in height (Hernandez and Mitsch, 2007), were inserted 10 cm deep into the sediment at each sampling site. Acetylene gas was injected into the chambers until 10% (v/v) of the headspace of the chamber was occupied by the gas. Then, headspace gas samples were collected every 20 min for 2 h. Accumulated nitrous oxide concentrations in the gas samples were analyzed using an electron capture detector (ECD) equipped gas chromatograph (GC-14A, Shimadzu) with a porapak-Q column. For calculating the denitrication ux, only the slope which showed a linear increase in nitrous oxide concentrations with time was selected. Two samples at the most were excluded in a 6 sample set when it was necessary to satisfy the signicance of linearity in the slopes. One data point from SM in September was eliminated from subsequent data analysis when it showed an extremely high denitrication rate, which was considered as an outlier. 2.3. Denitrifying bacterial community structure DNA from soil sample was extracted using an UltraClean soil DNA kit (MoBio) and kept at 20  C prior to analysis. A ngerprinting analysis using terminal-restriction fragment length polymorphism (T-RFLP) was conducted to determine denitrifying bacterial community structure. The 40 ml reaction mixture contained template DNA (approximately 100 ng), PCR buffer, dNTPs (nal conc. 200 mM), bovine serum albumin (8 mg), Taq polymerase (2.5 unit) (Promega, USA) and primer pair (each nal conc. 12.5 mM). The orescence-labeled primer pair nirS832F (GTN AAY GTN AAR GAR CAN GG) e nirS1606R-FAM (ACR TTR AAY TTN CCN GTN GG) was used as described by Liu et al. (2003). The PCR was performed using a PTC-100 thermal cycler (MJ Research, USA). A denaturation step was at 94  C for 2 min, followed by 35 cycles at 95  C for 30 s, primer annealing at 65  C for 60 s, and extension at 72  C for 1 min and the nal extension was performed at 72  C for 10 min. The PCR product was separated by electrophoresis and puried using a DNA purication kit (MoBio). Puried PCR products were then digested using the restriction enzyme HhaI (Promega) at 37  C for 6 h, followed by heat inactivation of the restriction enzyme at 65  C for 15 min. The analysis of T-RFs was performed using an ABI 3730 XL automatic sequencer (Applied Biosystems). 2.4. Quantication of denitrifying bacteria In order to quantify denitrifying bacteria in our soil samples, we used quantitative real-time PCR (RT-PCR) targeting the nirS gene.

Statistical analyses were performed using SPSS ver. 15.0. Signicant differences (P < 0.05) in obtained data within treatments and sites were determined according to two-way ANOVA, followed by Tukeys test. Stepwise linear regression was applied to determine the relationship between denitrication and chemical properties in wetlands. The denitrifying bacterial community structure and diversity were evaluated based on the number and area of terminalrestriction fragments (T-RFs) in each phase. Lengths with less than a 0.5 bp difference were considered to be the same peak. The peak area for each fragment was converted to a proportion of the total area in each community composition and represented as abundances of T-RFs. The bacteria diversity was calculated using the Shannon diversity index (Shannon, 1948). The differences in community structure between treatments were analyzed using multi-response permutation procedures (MRPP) and non-metric multidimensional scaling (NMS) in PC-ORD ver. 5.0 (McCune and Mefford, 1999). 3. Results 3.1. Responses of denitrication to hydrologic pulsing The water level drawdown and drying followed by the reooding event in the wetlands resulted in signicant changes in denitrication (Fig. 2). During the saturated period, denitrication rates decreased dramatically, except at the inow site. In contrast, denitrication rates increased during reooding to similar or slightly higher values than the original rates prior to the hydrologic pulsing. Interestingly, denitrication in the SM increased

800

Denitrification (g N2O-N m-2 hr-1)

Inundateda Saturatedb Dryingb Refloodinga

Site: P=0.547 Hydrology: P=0.001 Interaction: P=0.046

600

400

200

0 IN DM SM OUT

Fig. 2. Denitrication rates (mean SE) by hydrologic pulsing treatment in the experimental wetlands. IN, DM, SM and OUT stand for inow area, deepwater marsh, shallow water depth marsh and outow areas, respectively. Different letter next to the legend represents signicant differences among each period at P < 0.05 based on Tukey analysis in two-way ANOVA. Also, signicances of two-way ANOVA for denitrication rates in relation to different site, hydrologic pulsing, and their interaction are shown.

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K. Song et al. / Soil Biology & Biochemistry 42 (2010) 1721e1727 Table 2 Denitrication rates and chemical characteristics (mean SE) in response to hydrologic pulsing in created experimental wetlands at Olentangy River Wetland Research Park.a SUVA NO No. of gene Denitrication DOC 3 (mg N2OeN (mg g soil1) (L mg-M1) (mg g soil1) copy (1012) m2 h1) Inundated 301 32a Saturated 133 29b Drying 71 21b Reooding 271 42a 0.49 0.29 0.40 0.35 0.05 0.03 0.02 0.05 1.50 1.46 0.93 1.01 0.14 0.21 0.11 0.11 4.16 4.02 4.61 4.28 0.28 0.25 0.41 0.21 1.49 1.60 1.67 1.02 0.11a 0.13a 0.16a 0.09b

signicantly after reooding, resulting in 175% higher rates than during the original inundated period (Fig. 2). The magnitude of effect on denitrication rate due to hydrologic pulsing was in the order of SM > DM > OUT > IN based on the slope of each regression (Fig. 3). A signicant correlation was found between denitrication rates and water level changes (R2 0.31, P < 104) although the individual regressions from the outow and inow sites were not statistically signicant. Denitrication, soil DOC, SUVA, and NO 3 concentration and the nirS gene copy number for each hydrologic phase are presented in Table 2. SUVA exhibited lower values during drying and reooding periods. Nitrate concentrations in the soil were 4.74 and 4.22 mg g soil1 in drying and reooding periods, respectively, higher concentrations than those in the inundated and saturated periods. However, these chemical properties did not exhibit signicant differences among hydrologic pulsing phases (Table 3). Although DOC also did not show signicant differences among hydrologic pulsing periods, stepwise linear regression showed that denitrication rates could be explained by DOC concentration (R2 0.15, P 0.03, Fig. 4).

a Different letters next to the values indicate the signicant differences (P < 0.05) among different hydrologic periods by Tukey analysis in two-way ANOVA. SUVA represents specic UV absorbance of organic carbon.

3.2. Responses of denitrifying bacterial community structure and quantity to hydrologic pulsing The gene copy number exhibited signicant differences among hydrologic phases as well as sites (Fig. 5). Water level drawdown and the drying period led to a slight increase in the nirS gene copy number in all sampling sites except the inow area. Furthermore, sudden reooding led to a decrease in nirS gene copy number in every site. Shannon diversity index was also not affected by hydrologic pulsing (Table 2). However, the diversity indices between vegetated marsh and non-vegetated areas were signicantly different according to one-way ANOVA (P 0.048). The diversity indices of the marsh areas (2.42 0.07 for DM and 2.52 0.08 for SM) were higher than those of the non-vegetated areas (2.31 0.11 for IN and 2.26 0.12 for OUT). The community structure of denitrifying bacteria exhibited signicant differences among sampling sites while hydrologic pulsing did not induce any signicant differences of community structure among the inundated, saturated, drying and reooding periods (Table 3).

NMS ordination conrmed a distinct difference of community composition depending on the presence/absence of marsh vegetation rather than the effect of hydrologic pulsing treatments (Fig. 6). The rst 2 axes accounted for 54.7% and 39.3% of community differences. In addition, MRPP statistical A-values for T-RFs conrmed the signicance of differences between open water and vegetated marsh areas with an A-value of above 0.21 (P < 0.05, Table 3). 4. Discussion Hydrologic pulses such as the one described here are expected to occur more often and with higher intensity due to climate change. A decrease of denitrication with water level drawdown has been commonly expected and reported (e.g. Groffman and Tiedje, 1988; Baldwin and Mitchell, 2000). However, increased denitrication in lowered water level in peatland was also reported (Bandibas et al., 1994; Dowrick et al., 1999) and they suggested that denitrication peaked in saturated, not water logged conditions. Previous studies about effects of hydrologic pulsing on microbial communities have been generally focused on eubacteria and fungi (Fierer et al., 2003; Gordon et al., 2008; Fenner et al., 2005). For denitriers, only drought effects (Kim et al., 2008) and the effects of different hydrologic regime in different locations (Bougon et al., 2009) have been assessed. Therefore, the response of denitriers under hydrologic pulsing is still not known. In addition, the relationship among denitrifying bacterial community, quantity and their process rates have not been considered in most previous studies, even though the altered denitrication rate could be a result of physiological response to hydrologic pulsing of denitrifying bacterial communities (Schimel and Gulldege, 1998; Schimel et al., 2007; Bougon et al., 2009). In this study, we had hypothesized that the responses of denitrication rates in wetlands to hydrologic pulsing would be induced by a shifted denitrifying bacterial community. However, we found that the denitrication process and denitrifying bacterial community responded differently to hydrologic pulsing events in wetlands, which did not match with our hypothesis.

Denitrification (g N2O-N m-2 hr-1)

600

IN (a= 8.4) DM* (a= 18.8) SM* (a= 33.3) OUT (a=15.2) Total* (a= 16.4)

400

200

Table 3 MRPP A-values of denitrifying bacterial community structure for different hydrologic and site effects in the wetlands (*P < 0.05, **P < 0.01).
0 -0.10

Hydrology
-0.05 0.00 0.05 0.10 0.15

A-value Saturated Drying Reooding Drying Reooding Reooding 0.001 0.042 0.013 0.006 0.014 0.008

Site IN DR SR OUT SR OUT OUT

A-value 0.25* 0.31** 0.00 0.00 0.21** 0.25**

Water table level (m)

Fig. 3. The responses of denitrication rates depending on surface water level changes by hydrologic pulsing in each sampling site using linear regression. Slope a (102) of each regression line is shown in the legend. An asterisk shows the signicance of regression at P < 103. Averaged denitrication rates and water levels from each sampling site in the wetlands were used.

Inundated

Saturated Drying

DR SR

K. Song et al. / Soil Biology & Biochemistry 42 (2010) 1721e1727

500 DeN=346.7 DOC+61.2 (R2=0.147, P=0.03, n=44) 400

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Denitrification (g N2O-N m-2 hr-1)

300

200

100

0 0.0 0.2 0.4 0.6 0.8 1.0

DOC (mg g soil-1)

Fig. 4. Denitrication rates in relation to DOC concentration in the wetlands by stepwise linear regression model. Fig. 6. Non-metric multidimensional scaling (NMS) ordination of denitrifying bacterial community determined by nirS T-RFLP prole. Points with different shapes represent different hydrologic treatment samples with an averaged proportional abundance of T-RFs.

4.1. Responses of denitrication to hydrologic pulsing Denitrication decreased with drying and increased during the reooding period. This result is consistent with previous results, which showed a major inuence of hydrologic changes on denitrication rates (e.g. Groffman and Tiedje, 1988; Freeman et al., 1997; Goldberg and Gebauer, 2009). Freeman et al. (1997) also observed a nitrate release from peatland soils with a decline of N2O emission due to lowered denitrication rate over a drying period and the reversible responses when the inow was re-initiated. In addition, the extent of the changes in denitrication rates under hydrologic pulsing was spatially different within sampling locations. For example, the highest denitrication rate during the reooding period was exhibited in the shallow marsh site (SM) although SM sites exhibited the lowest rates during the inundated period. This result could be explained by the magnitude of water level change as well as higher microbial activity in the vegetated marshes. SM sites maintained a relatively longer drying period due to low water level; this could have led to inorganic nutrient release by aerobic decomposition. Release of nitrate in dried peatlands

(Freeman et al., 1993), enhanced extracellular enzyme activities and the following large ush of inorganic nutrients after drying period in wetlands (Song et al., 2007) have been reported. Therefore, released inorganic nutrients during a long drying period could result in the drastic increase in denitrication by increasing substrate availability in the SM site. DOC also exhibited a positive correlation with denitrication, indicating that high denitrication is associated with high carbon availability. Therefore, our result suggests that denitrication is regulated by physicochemical characteristics such as substrate availability and water level in wetlands.

4.2. Responses of denitrifying bacterial community structure and quantity to hydrologic pulsing Different from the response of denitrication rate, hydrologic pulsing had no distinct inuence on the community structure of denitrifying bacteria in the wetlands. One possible explanation involves the physiological characteristics of denitriers. Most denitriers are facultative, which prefer oxygen instead of nitrate as an electron acceptor (Zumft, 1997). Therefore, the changes in redox condition resulting from hydrologic pulsing might not have a signicant inuence on denitriers. Another possibility is that microbial communities, that experience repeated mild stresses, tend to be more resistant to stress than other communities that have not experienced stress (Fierer and Schimel, 2002; Grifths et al., 2005; Schimel et al., 2007; Philippot et al., 2008). Reduced microbial mortality rate by repeated drying-reooding events (Fierer and Schimel, 2002) and no signicant changes in denitrifying bacterial community structure under drought in natural riparian ecosystems (Kim et al., 2008) have been observed. Therefore, we speculate that water stress-tolerating denitriers could be selected by natural succession and competition as the created wetlands have undergone natural drying and reooding events. While hydrologic pulsing did not affect denitrifying bacterial communities, we observed large differences in denitrifying bacterial community structure in the presence/absence of vegetation. The changes in denitrifying community structures in response to their habitat conditions like temperature, DO gradient, vegetation or land use type have been reported (Avarahami et al., 2002; Rich et al., 2003; Castro-Gonzlez et al., 2005). Nutrients or soil type

Inundated 30

ab

Saturated a Drying a Reflooding

b

Number of Gene Copy (1011)

Site: P=0.043 Hydrology: P=0.001 Interaction: P=0.095

20

10

0 IN DM SM OUT

Fig. 5. The patterns of nirS gene copy number (mean SE) with hydrologic pulsing events from different sampling sites in the experimental wetlands. Different letter next to the legend represents signicant differences among each period at P < 0.05 based on Tukey analysis in two-way ANOVA. Also, signicances of two-way ANOVA for denitrication rates in relation to different site, hydrologic pulsing, and their interaction are shown.

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appeared to have greater impacts on microbial communities (Lundquist et al., 1999; McLean and Huhta, 2000; Fierer et al., 2003; Bossio et al., 2006). Bossio et al. (2006) reported that the differences of microbial community structure between sampling sites was greater than seasonal variability within each site. Different vegetation types can also inuence the denitrifying bacteria communities (Bremer et al., 2007). However, since two wetlands in this study have relatively similar vegetation communities, dominated by Typha spp. and taxonomically similar species, we could not nd the effect of different vegetation type on microbial community structure. Therefore, our results, along with previous studies, support the hypothesis that microbial community structure is inuenced by long-term physicochemical characteristics rather than by episodic hydrologic disturbances. We originally expected that reduction in denitrifying bacterial quantity by physiological stress under hydrological pulsing would restrict denitrication rate. In this study, nirS gene copy number slightly increased or remained constant during the drying period, and it dropped as reooding occurred. We suggest that while facultative denitriers can survive in aerobic condition, reooding caused serious osmotic stress (Schimel et al., 2007), resulting in a decline of gene copy number. This change in quantity of denitriers did not correspond with the response of denitrication rates under hydrologic pulsing. For example, the denitrication rate increased while denitrifying bacterial quantity decreased during reooding period. This result was possibly because the nirS gene copy number does not represent the number of active denitrifying bacteria (Philippot and Hallin, 2005). This kind of discrepancy between microbial biomass and process rates has been reported (Pesaro et al., 2004; Gordon et al., 2008). Gordon et al. (2008) showed the decrease of microbial biomass C and N with an increase of basal respiration as a result of drying and reooding events. Pesaro et al. (2004) also reported that while respiration rates were recovered, soil microbial biomass was not after drought. Some studies have suggested that different denitrifying bacterial community composition or their quantity would contribute to denitrication rates or N2O emissions due to their physiological differences in communities (Cavigelli and Robertson, 2000, 2001; Rich et al., 2003). However, the different responses of denitrication and denitrifying bacterial communities to hydrological pulsing events in this study imply that the community structure was not a determinant of the denitrication rates. This result supports the insurance hypothesis, suggesting a functional redundancy of biodiversity (Yachi and Loreau, 1999; Loreau et al., 2001).

Acknowledgements H. Kang is grateful to AEBRC, EcoSTAR, EcoRiver and NRF (20090079549) for nancial support. This research is also partially supported by the Olentangy River Wetland Research Park and by the U.S. Environmental Protection Agency grant (EM83329801-0 and MX95413108-0). References
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4.3. Conclusions This study showed that short-term hydrologic pulsing has a major inuence on denitrication rates in wetlands. This change in denitrication was not induced by the response of denitrifying bacterial community to hydrologic pulsing. Instead, DOC and water level could initially drive the changes in denitrication rates in the short-term hydrologic disturbance. While hydrologic pulsing did not induce any signicant differences in denitrifying bacterial community structure, the presence and absence of vegetation did inuence denitrifying bacterial community structure. Our results suggest the prevailing importance of physicochemical properties affecting denitrication rate over community composition or quantity of denitrifying bacteria. However, the abundance of denitrifying bacteria was reduced by hydrologic pulsing, particularly by the reooding event. Therefore, repeated and severe drying-reooding events with climate change over the long term could induce large decreases of denitrifying bacteria which could result in poorer water quality in the wetlands and adjacent rivers.

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