Changes in Denitrification Potentials and Riverbank Soil Bacterial Structures along Shibetsu River, Japan

Research Faculty of Agriculture, Hokkaido University, Kita 9 Nishi 9 Kita-ku, Sapporo, Hokkaido 060-8589, Japan Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9 Kita-ku, Sapporo, Hokkaido 060-8589, Japan Graduate School of Environmental Science, Hokkaido University, Kita 9 Nishi 9 Kita-ku, Sapporo, Hokkaido 060-8589, Japan School of Agriculture, Hokkaido University, Kita 9 Nishi 9 Kita-ku, Sapporo, Hokkaido 060-8589, Japan


Introduction
Riverbank ecosystems are very important ecological zones in relation to nitrogen (N) cycle.Nitrogen (N) can be a source of water pollution particularly with excess nitrate (NO 3 − -N) derived from agricultural systems.During the filtration process performed by riverbank ecosystems, the concentration of NO 3 − -N may decrease due to the gasification of NO 3 − -N [1].Denitrification is one of the microbial processes responsible for this gasification process. is process reduces NO 3 − -N to gases such as nitrous oxide (N 2 O) and di-nitrogen (N 2 ).us, it is important to evaluate the factors controlling denitrification potential in riverbanks along river ecosystems.
While denitrification can potentially reduce NO 3

−
-N from the riverbank ecosystems, it can also negatively impact the environment because N 2 O is a greenhouse gas.e global warming potential of N 2 O is reported to be 298 times greater than carbon dioxide (CO 2 ) [2]. us, previous studies concluded that the riverbank ecosystems' denitrification can increase the environmental risk of N 2 O emissions [3,4].Nitrification process (the oxidation of ammonia to NO 3 − -N) is also an important N 2 O-producing process when soils are well aerated [5], but denitrification is more important when soil is wetter or submerged with water [6].Carbon and N contents in soils and the amount of readily available inorganic-N positively control the denitrification potentials because these are substrates for the activity of denitrifying microbial communities [7,8].Soil pH also controls denitrification potentials although the relationship between pH and denitrification is markedly influenced by other factors such as the size of denitrifying microbial communities [9,10].Also, temperature, oxygen availability, and physical factors such as hydraulic conductivity of the sediment were reported as controlling factors of denitrification potentials in riverbank ecosystems [3,[11][12][13].However, a review article suggested that further studies are needed in this area particularly using three-dimensional approaches (spatial heterogeneity, soil depth, and time courses) [14].
To further understand the factors controlling the variability of denitrification potentials in riverbank ecosystems, microbial approaches are useful.Denitrification processes are mainly performed by a group of microbes called denitrifiers.Microbes capable of denitrification are phylogenetically extremely diverse [15].For example, many groups in the phylum Proteobacteria, such as auera, Paracoccus, Hyphomicrobium and Comamonas, were reported to be performing denitrification in a wastewater system [16].Also, denitrifying microbes interact with other microbes in soils.In recent years, 16S rRNA-based bacterial community structural analyses in natural ecosystems, including riverbank ecosystems, are becoming more common [17].us, 16S rRNA approaches may be useful to identify potential denitrifiers in riverbank ecosystems and to identify factors controlling the variability in denitrification potentials.Similar approaches have already taken to identify denitrifying microbial communities in agricultural soils [18,19].For example, Ishii et al. [19] performed 16S rRNA analyses of soil DNA extracted from a paddy field and identified several bacterial genus, including Herbaspirillum in the phylum Proteobacteria, as key players during denitrification process.However, few studies compared multiple sites using the 16S rRNA approaches to find out whether the same bacterial species are controlling the magnitudes of denitrification or not.us, by compiling the heterogeneity of denitrification potentials in different riverbank ecosystems along a river with basic soil characteristics (i.e., soil pH, soil carbon, moisture, and texture) and 16S rRNA, we may be able to identify microbial phyla/families controlling the magnitude of denitrification potential of a soil in a larger scale.e information can be important to evaluate ecosystem disservices of rivers, particularly N 2 O emissions.Rivers located in an area with developing farming industry and/or with large cities may require this type of information to develop sustainable future plans.
us, in this study, we sampled riverbank soils along Shibetsu River in Hokkaido, Japan, and measured their denitrification potentials along with other chemical properties.Shibetsu area is known as one of the most developed dairy industry zones in Japan and N-related water pollution is an issue in this area [20].We hypothesized that the variability of the denitrification potentials in riverbank soils along Shibetsu River can be explained by the presence/absence of specific bacterial groups.Also, we hypothesized that the chemical characteristics of the riverbank soils partly influence the magnitudes of denitrification potentials.

Riverbank Soil Sampling.
Riverbank soil was sampled in September 2016 at eight sites across the Shibetsu River basin, Hokkaido (Figure S1, Table S1).We sampled the soils as evenly as possible along the river but sampling was not possible when the area was managed as private farms (e.g., between E and F). e sites B and G were in stream tributaries to the Shibetsu River.Between the sampling points F and G, Nakashibetsu city is located, and most of the riverbank was solidified in concrete; thus, we did not sample the soil in the city.Soil sampling has performed using a soil auger, and topsoil (0-15 cm depth) and subsoils (15-30 cm depth) were taken separately.For each sampling point, 2-4 soil cores were taken from the 1 m × 1 m zone.e samples were then mixed and placed in a plastic bag.e sampled soils were placed in plastic bags and stored at <4 °C until analyses.e basic soil characteristics (pH, ammonium-N, nitrate-N, total C, and total N) were listed in Table S2.e soil type for the sampling points A to F was immature sandy Fluvisol.For the sampling points G and H, the soil was still Fluvisol, but it was not an immature soil.In the vicinity of the sampling points G and H, there was a large peatland zone, but along the Shibetsu River, alluvial deposits formed Fluvisol and our sampling depth (up to 30 cm) did not observe any peat materials [21].At the sites B, E, and F, we could not sample the subsoils because soils only developed shallowly and rocks were present below 15 cm.e annual rainfall in this area averaged 1158 mm. e average temperature was 5.4 °C.

Riverbank Soil Characteristics.
For the riverbank soils at each site, soil moisture contents were measured by ovendrying the soil samples at 105 °C for >24 h.For pH water , fresh soil and MilliQ water were mixed in the ratio of 1:2.5, and pH was measured using a pH sensor (AS800, AS ONE Co., Japan).For pH KCl , fresh soil and 10% KCl were mixed in the ratio of 1 : 5, and pH was measured using the same pH sensor.e pH water refers to the acidity of the soil solution whereas the pH KCl refers to the acidity in the soil solution plus the research acidity in the colloids.e total C and total N were measured using 2400 Series II CHNS/O Elemental Analyzer System (PerkinElmer, Inc., Waltham, USA).e inorganic-N concentrations (ammonium-N (NH 4 + -N) and NO 3 − -N) were measured by extracting the fresh soil with 10% KCl and filtering through 1 μm (No. 5C, Toyo Roshi Kaisha, Ltd., Japan).

Denitrification Potentials and eir Relationships with
Soil Characteristics.Denitrification potentials for the soil samples were measured using an acetylene block method [23] under three different conditions, (1) without any substrate additions, (2) with added NO 3 − -N, and (3) with added NO 3 − -N and glucose-carbon (glucose-C) with three replicates per sample.is experiment was performed under anaerobic conditions (>99% N 2 ).First, approximately 2.5 g of fresh soil was placed in 100 ml glass bottle and one of the three solutions, (1) 5 ml of MilliQ water, (2) 5 ml of 0.72 g NO 3 − -N•l −1 , or (3) 5 ml of 0.72 g NO 3 − -N and 0.5 g glucose-C•l −1 .en, the bottles were capped using septa and aluminium caps, and their headspaces were replaced with >99%

2
Applied and Environmental Soil Science N 2 using two needles (one to supply N 2 and another to evacuate atmospheric air).Approximately 10% of the headspace of each 100 ml bottle was replaced with acetylene and the bottles were incubated for 2 hrs at 25 °C.e bottles were gently shaken during the incubation.After the incubation period, 30 ml of the gas inside of each bottle was sampled using a syringe and placed in an evacuated vial for the measurement using a gas chromatograph equipped with an electron capture detector.e denitrification potential for each soil was then expressed as e increase of the denitrification potentials due to the addition of glucose-C and NO 3 − -N was evaluated using a multiple regression model to investigate the best fitted model, and the smallest Akaike Information Criterion (AIC) was calculated. is approach was commonly used to minimize the number of parameters (soil characteristics) to explain the variables (denitrification potentials).
e multiple and adjusted R 2 values were 0.582 and 0.500, respectively.Based on the AIC analyses, the following model was established: (1)

16S rRNA Bacterial Community Structures.
For the topsoil samples (0-15 cm, eight samples in total), we investigated the changes in 16S rRNA bacterial community structures before and after the anaerobic incubation with NO 3 − -N and glucose-C, with two replications (eight soils × before and after C and N addition × two reps � 32 samples).
e incubation procedure and substrate concentrations were the same as the method used for the measurement of denitrification potential except that the incubation length was 48 h, and the number of replication was two for this 16S rRNA experiment.
For the soil samples before and after the anaerobic incubation, soil DNA was extracted using PowerSoil Kit (MO BIO Laboratories, Inc. Carlsbad, USA).
After the amplification, the PCR products were analysed using a next-generation sequencer (Ion PGM, ermo Fisher Scientific Inc.).
e obtained sequence data were analysed using the software QIIME [24].ere were eight soil samples, and we analysed the bacterial community structures before and after the addition of NO 3 − -N and glucose-C in duplicates.us, we had 32 samples in total.Using QIIME, we obtained the community structure data with taxonomies, at phylum, order, family, and genus levels.

Statistics.
For the denitrification potential data, two-way ANOVA with F test was performed to investigate the effect of sampling sites and the treatments (the addition of NO 3 − -N and glucose-C).Post hoc Tukey's test was performed when there was a significant interaction between the sampling sites and the treatments (p < 0.05) to evaluate the effect of treatments for each sampling site.
en, the relationship among the denitrification potentials (with NO 3 − -N and glucose-C) and other soil characteristics was investigated using linear regressions.
For the increase in denitrification with added C and N (difference between the denitrification potential with C and N, and the denitrification without any substrate addition), we performed a multiple regression with other environmental variables.e environmental variables were pH water , pH KCl , NH 4 + -N, NO 3 − -N, total C, and total N. To determine the best fitted model based on the multiple regressions, we used the Akaike's Information Criterion (AIC) approach.Using this approach, we aimed to simplify the model using minimum number of the variables.e smaller the AIC, the better the model is, according to the AIC approach.
For the soil microbial community structure data, significantly increased phyla after the anaerobic incubation with added C and N were identified using t-tests.For the significantly increased phyla, they were further studied more in details (at the class/order/genus levels).Also, for the phylum level community data, canonical-correlation analysis (CCA) was performed to investigate the similarity among the communities.e statistical analyses were performed using the software RStudio (version 1.1.383).
e riverbank soil pH water varied from 5.53 to 6.82 and pH KCl varied from 4.27 to 6.43; thus, it was slightly acidic.

Denitrification Potentials and eir Relationships with
Soil Characteristics.In the topsoil, the denitrification potentials were generally higher with added C and N, when compared to those without any added substrates (Figure 1(a)).
Both sampling sites and the addition of C and N had significant effects on the denitrification potentials (both resulted in p < 0.001).Also, there was a significant interaction between the sampling sites and the treatments (p < 0.001).For the site A and B, the denitrification potential did not respond to the addition of N but increased with the addition of C and N.For the sites C, D, and H, both the addition of N and addition of C and N resulted in higher denitrification potentials when compared to the controls.e sites E, F, and G did not clearly respond to the addition of substrates.
In the subsoil, the addition of C and N also significantly increased the denitrification potentials, when averaged across the sampling sites, but the significance was relatively weaker (p < 0.05) when compared to the topsoil (Figure 1(b)).e sampling sites also had a significant influence on the Applied and Environmental Soil Science denitri cation potentials in the subsoil too (p < 0.01), but there was no interaction between the sampling sites and the treatments.e site C had relatively higher denitri cation potentials when compared to other subsoils, but the variability among the replications was also larger when compared to other subsoils.
e denitri cation potentials with added C and N correlated positively to the soil moisture at the sampling (p < 0.001) and correlated negatively to soil pH water (p < 0.01) (Figures S2(a

e E ect of C and N Addition (Increase in Denitri cation) and Environmental Variables.
e increase in denitri cation with substrates (denitri cation potential with C and N minus denitri cation potential without any substrate addition) was negatively correlated with pH water , when all parameters' correlations were tested (Figure S3).Based on the multiple regression model, the smallest AIC was achieved when we used pH water and NH 4 + -N as the environmental parameters (1).

16S rRNA Bacterial Community
Structures.Soil microbial community structures were measured before and after the addition of C and N. e di erence was characterized by an increase in Proteobacteria, at the phylum level, from 44 ± 8% (before) to 65 ± 14% after the addition of C and N, when averaged across the sampling site (error s.d.) (Figure S4).
When the changes within Proteobacteria phylum (class and order levels) were compared before and after the incubation with C and N, the major increase of this phylum was supported by the increase of the class Gammaproteobacteria from 9.3 ± 8.5% (before C and N addition) to 27 ± 15% (after C and N addition) (Figure S5).Within Gammaproteobacteria, the increase of an order Aeromonadales was conspicuous (relative abundance within the whole bacterial community 0.0 ± 0.0% and 17 ± 18%, without and with C and N addition, resp.)(Figure S6).Within the order Aeromonadales, the genus Aeromonadaceae Tolumonas was dominated (relative abundance within the whole bacterial community 0.0 ± 0.0% and 16 ± 17%, without and with C and N addition, resp.)(Figure 2).

Denitri cation Potentials and Soil Microbial Community
Structures.We have not found a clear relationship between the 16S rRNA bacterial community data and the magnitudes of the denitri cation potential.e results from the CCA analyses suggested that sites B, C and E showed minor changes in their community structures whereas other sites showed more signi cant and similar changes in their community structures, after the addition of C and N (Figure S7).However, the magnitudes of the community structure changes due to the addition of C and N and the denitri cation potentials were not related.Also, at the genus level, the magnitudes of the increase in Aeromonadaceae Tolumonas (Figure 2) were compared to the denitri cation potentials in the topsoil (Figure 1(a)) but there was no signi cant relationship between them.

Denitri cation Potentials and eir Relationships with Soil
Characteristics.
e denitri cation potentials were higher with added glucose-C and NO 3 − -N, when compared to those FIGURE 1: Denitri cation potentials for the topsoils (0-15 cm) and subsoils (15-30 cm).Small letters de ne signi cant di erences among three substrate treatments ("control," "with N," and "with C and N"), for each sampling site (A-H), for the topsoils.For the subsoils, the small letters de ne signi cant di erences among the di erent sites.e error bars were standard deviations (n 3).(a) Topsoil and (b) subsoil n.s.= not signi cant, * p < 0.05, * * p < 0.01, * * * p < 0.001.

4
Applied and Environmental Soil Science with added NO 3
e range of the denitri cation potentials without C and N was also similar to the previous study measuring denitri cation potentials in the same river [29].
e increased rates of denitri cation potentials with the addition of glucose-C and NO 3 − -N, compared to the denitri cation potentials without the addition of C and N, markedly varied among the samples (Figure 1).It is di cult to fathom reasons behind these variations with our limited sample numbers; however, this phenomenon is important to understand the factors controlling the gaseous loss of N from this ecosystem, including ecosystem disservice such as N 2 O emissions.A previous study concluded that the denitri cation potentials in a riverbed sediment (South Platte River Basin, Colorado, US) positively responded to the addition of acetate-C (ranging from 0 to 650 μmol•l −1 ), but the response rates decreased with increasing sampling depth (sampling depth 0.61, 1.22, and 2.44 m). e current study similar results with shallower depth range (0-15 and 15-30 cm) with multiple sites because the shallower soils (Figure 1(a)) clearly responded to the addition of NO 3 − -N and glucose-C + NO 3 − -N, when compared to the deeper soils (Figure 1(b)).us, even along the same river, the impact of nutrient loading on denitri cation and limiting factors of denitri cation potentials can be variable.e movement and mixture of ground and surface water have been reported as a factor controlling the variability of denitri cation potential in Mississippi river [30].Also, di erent plants have reported to have di erent impacts on denitri cation potentials in their root zone soils [31].
us, the vertical and lateral movements of water and vegetation types may explain some of the variabilities in denitri cation potentials found in the current study [32].Further studies in this area may reveal some key parameters to predict the impact of nutrient loading on the riverbank area, and the parameters may be useful to plan future land use along a river.
Denitri cation potentials with added C and N correlated positively to the soil moisture at the sampling (p < 0.001) and correlated negatively to soil pH water (p < 0.01) (Figures S2(a -N) contents were not correlated to the denitri cation potentials with glucose and KNO 3 (Figures S2(c)-S2(f)).us, in our experiment, the soil pH and moisture condition relatively more strongly controlled the denitri cation potentials than the nutritional condition (total C, total N, and inorganic N).Moisture status in soils was previously reported as main factors controlling soil denitri cation potentials in the riparian area [33,34].For example, a previous report stated that with increasing soil moisture from 15% to 65% (gravimetric), denitri cation potentials of the riparian zone soils sampled in Gwynns Falls Watershed, Baltimore, US, linearly increased from approximately zero to 500 ng•N•g −1 •h −1 [33]. is relationship was similar to what we found in the current study although the moisture range was narrower (30-55%) (Figure S2(a)).
Soil pH negatively correlated to denitri cation potentials in the current study (pH ranged approximately 5.5 to 7.0, Figure S2(b)).A similar trend was observed when soil pH was compared with the increase in denitri cation potentials after the addition of C and N substrates (Figure S3).Contrastingly, Čuhel et al. [35] reported a positive correlation between soil pH and denitri cation potentials in grassland soils (pH ranged 5.5 to 7.7).
ere was another report showing pH did not in uence the denitri cation potential in soils (pH ranged 5 to 8) [32].In the study by Čuhel et al. [35], denitri cation potentials ranged between 200 and 1200 ng•N•g −1 •hr −1 .e range was similar in the study by Liu et al. [36] (200 to 2000 ng•N•g −1 •hr −1 ).However, the maximum denitri cation potential in the current study was 170 ng•g −1 •hr −1 , relatively smaller than the previous reports focusing on the e ect of pH on denitri cation potentials (Figure S2(b)).e sampling areas in the current study are often ooded; thus, a limited number of denitrifying microbes might be adapted to this area, compared to the soils tested in the previous studies.is might be a reason for the contrasting response of denitri cation potentials to pH, although further studies are needed.Also, historical pH changes were reported to be more important than shortterm changes of pH as a controlling factor of denitri cation potentials [37].We note that our soil sampling was one-o , and we do not know temporal changes in soil pH over seasons or years.Also, it is still uncertain why pH varies along the river.Previous studies showed that the land-use change in the area surrounding a river and vegetation types influenced the pH in riverbed area, and the vegetation types were determined due to seasonal flooding events [38,39].Further studies are needed in this area, along Shibetsu River.Soil NH 4 + -N levels were shown as an important factor controlling the magnitudes of the increase in denitrification potentials after the addition of glucose-C and NO 3 − -N, in the current study (1).It is difficult to fathom reasons behind this, but the soils tested in the current study had relatively higher amount of NH 4 + -N when compared to NO 3 − -N.In the current study, the in denitrification potentials after the addition of glucose-C and NO 3 − -N was tested under an anaerobic condition, thus nitrification-derived N 2 O was likely to be minor.Possibly, high soil NH 4 + -N levels suggest high mineralization of organic-N and relatively higher soil microbial activities, increasing the activity of denitrifiers.
At the field level, the amount of NH 4 + -N might be a limiting factor of denitrification because NH 4 + -N provides substrate (NO 3 − -N) for denitrifiers via nitrification.Nitrous oxide emissions are observed both from nitrification and from denitrification; thus, both processes can be a cause of environmental impacts from the riverbed system.

16S rRNA Bacterial Community Structures.
e difference between the soil microbial community before and after the C and N addition was characterized by a marked increase in Proteobacteria, at the phylum level (Figure S4).However, for the soils C and E, the changes in their bacterial communities due to C and N addition were relatively minor compared to other soils.Addition of C sources and the consequent increase in the phyla Proteobacteria in soils had been previously reported [27]; thus, Proteobacteria had been recognized as a fast grower responding to an addition of readily available substrates.
When the changes within Proteobacteria phylum (class and order levels) were compared before and after the incubation with C and N, the major increase of this phylum was supported by the increase of the class Gammaproteobacteria (Figure S5) and an order Aeromonadales (Figure 2).e class Gammaproteobacteria is known as one of denitrifier groups thus might be an important group to determine the capacity of rivers to remove excess-N.Another previous study also showed that Gammaproteobacteria Enterobacteriaceae was the dominate family when glucose was added to a forest soil under anaerobic conditions [28].
e order Aeromonadales is known as aquatic inhabitants in fresh water [29] and has a denitrifying capacity (reduction of NO 3 − -N to nitrite) [30].Also, G. Aeromonadales Tolumonas lignolytica was reported as a genus which utilizes lignin as a sole C source, and it was isolated from a tropical soil [31].

Linking Denitrification and Bacterial Community
Structures.In this study, we have not found a clear relationship between the changes in the 16S rRNA bacterial community structures before and after an anaerobic incubation with glucose-C and NO 3 − -N and the changes in the denitrification potentials (Figure 1, Figure S7).Contrastingly, there have been a few previous papers described a specific genus responsible for denitrification activity in specific conditions.For example, Betaproteobacteria Burkholderiales Oxalobacteraceae Herbaspirillum was reported to be a key player of denitrification activity in rice paddy soil under anaerobic condition with added C and N [19].ey tested the variabilities in soil's bacterial community structure and denitrification potentials using one rice paddy soil (with different regimes of C and N additions).us, they did not compare soils from multiple locations like the current study.
e relationship between the bacterial community structure and denitrification potentials may be site specific.A similar experiment using a soil sampled from a potato (Solanum tuberosum L.) field reported that Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas was the majority of the isolated denitrifiers [18].However, this study was also observing one soil from one farm and they used isolated bacterial communities on nutrient agar.us, the direct comparison of this study and the current study may be difficult.Another experiment using an arable soil suggested that the majorities of isolated denitrifiers were Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia, Pseudomonas, Gammaproteobacteria Xanthomonadales Xanthomonadaceae, Bacilli Bacillales Bacillaceae, and Actinobacteria Streptomycetales Streptomycetaceae [15]. is study also observed isolated denitrifiers on nutrient agar, unlike the current study, but we note that many of the previous studies highlighted a group of Gammaproteobacteria as a key player in denitrification process.
Our finding suggested that during the optimum condition for denitrification, a specific genus of bacteria may increase in a specific section of the Shibetsu river ecosystem.
is was based on the increase in Gammaproteobacteria Aeromonadales, observed after the incubation of the sampled soils with glucose-C and NO 3 − -N (Figure S6). e short-term (48 h) incubation of soils with glucose-C and NO 3 − -N stimulates the growth of fast-growing bacteria and Gammaproteobacteria is known as an extremely fastgrowing group although its abundance in natural soils is normally low when substrates are not available [40].However, our data suggested that although the specific family of bacteria increased with C and N, it is not necessarily they correspond to the magnitude of the increase in the denitrification potential.Gammaproteobacteria is also known as a metabolically flexible group of bacteria; thus, they perform denitrification only when conditions are favourable.For example, the lack of phosphorus is known as a factor controlling the denitrification performance of a group of Gammaproteobacteria [41].us, in future, we should observe the availability of nutrients other than C and N to fully understand the factors controlling the denitrification potentials of riverbank soils.Our study observed the whole bacterial community including nondenitrifying bacteria.Contrastingly, a previous study focused on soil DNA related to the function of denitrification enzymes (nirK, nirS, and nosZ genes or nitrite and nitrous oxide reductase encoding genes, resp.)[42].
ey found that the microbial communities harbouring these genes were responsible for denitrifier-derived nitrous oxide emissions from a waterlogged soil.us, one 6 Applied and Environmental Soil Science limitation of our study is the lack of information on the relationship between the whole bacterial community structures and the bacterial community-harbouring denitrification genes.A further limitation of our study is also due to the lack of information about the amount of bacterial biomass.Future studies should focus the relationship between the increase of bacterial biomass and denitrification potential.Additionally, the numbers of our sampling points and replication to analyse DNA were relatively small to discuss the detailed mechanisms behind the relationship between denitrification and soil bacterial community structures.Further studies are needed in larger scale with more detailed samplings.

Conclusion
Denitrification potentials' variability along the riverbank soils of Shibetsu river, Hokkaido, Japan varied from 7.43 to 181 μg•N•g −1 •h −1 with added glucose-C and nitrate-N substrates.Added C and N increased the denitrification potentials in some soils but not for others.ere was a negative relationship between the soil pH and the denitrification potential; thus, further investigation is needed to understand the mechanisms controlling the pH of riverbank soils.Total C, N, and inorganic-N contents in the sampled soils did not correlated to the denitrification potentials.e soil bacterial community data, based on the 16S rRNA analyses, taken before and after the anaerobic incubation with added C and N suggested that a bacterial family Gammaproteobacteria Aeromonadaceae Tolumonas markedly increased in most of the riverbank soils we sampled.However, the magnitudes of the increase in G. Aeromonadaceae Tolumonas were not related to the denitrification potentials.Further studies are needed to understand the microbial factors controlling the denitrification across a river since riverbank soil often provides an important ecosystem service to remove excess N.

Figure 2 :
Figure2: Changes in relative abundance of classes within the order Aeromonadales (within Proteobacteria Gammaproteobacteria), based on 16S rRNA analysis before and after the addition of C and N. e capitals A to H de ne the sampling sites.e word "before" describes the data before the addition of C and N whereas "with C and N" suggested the data from the soils with added C and N.