Response of Spatial Patterns of Denitrifying Bacteria Communities to Water Properties in the Stream Inlets at Dianchi Lake, China

Yi, Neng; Gao, Yan; Zhang, Zhenhua; Wang, Yan; Liu, Xinhong; Zhang, Li; Yan, Shaohua

doi:https://doi.org/10.1155/2015/572121

International Journal of Genomics

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusions Acknowledgments References Copyright Related Articles

Special Issue

Environment-Living Organism’s Interactions from Physiology to Genomics

View this Special Issue

Research Article | Open Access

Volume 2015 | Article ID 572121 | https://doi.org/10.1155/2015/572121

Response of Spatial Patterns of Denitrifying Bacteria Communities to Water Properties in the Stream Inlets at Dianchi Lake, China

Neng Yi,^1,2Yan Gao,^1,2Zhenhua Zhang,^1,2Yan Wang,^1,2Xinhong Liu,^1,2Li Zhang,^1,2and Shaohua Yan^1,2

Academic Editor: Marián Brestič

Received03 Jul 2015

Accepted16 Aug 2015

Published04 Oct 2015

Abstract

Streams are an important sink for anthropogenic N owing to their hydrological connections with terrestrial systems, but main factors influencing the community structure and abundance of denitrifiers in stream water remain unclear. To elucidate the potential impact of varying water properties of different streams on denitrifiers, the abundance and community of three denitrifying genes coding for nitrite (nirK, nirS) and nitrous oxide (nosZ) reductase were investigated in 11 streams inlets at the north part of Dianchi Lake. The DGGE results showed the significant pairwise differences in community structure of nirK, nirS, and nosZ genes among different streams. The results of redundancy analysis (RDA) confirmed that nitrogen and phosphorus concentrations, pH, and temperature in waters were the main environmental factors leading to a significant alteration in the community structure of denitrifiers among different streams. The denitrifying community size was assessed by quantitative PCR (qPCR) of the nirS, nirK, and nosZ genes. The abundance of nirK, nirS, and nosZ was positively associated with concentrations of total N (TN) and (). The difference in spatial patterns between nirK and nirS community diversity, in combination with the spatial distribution of the nirS/nirK ratio, indicated the occurrence of habitat selection for these two types of denitrifiers in the different streams. The results indicated that the varying of N species and together with pH and temperature would be the main factors shaping the community structure of denitrifiers. Meanwhile, the levels of N in water, together with , tend to affect the abundance of denitrifiers.

1. Introduction

It has been commonly observed that a large but variable proportion of aquatic N removal occurred in freshwater ecosystems, including groundwater, streams, lakes, and wetlands [1, 2]. The variation in microbial abundance and community structure among different aquatic ecosystems has been recognized as one of the most important factors which contributed to the changing of N biogeochemical cycling process in different aquatic ecosystems [3, 4]. Therefore, studying the spatial patterns of functional microbial guilds can help us to understand the relationships between microbial community ecology and the related ecosystem functions. Canonical denitrification has been generally considered as the main mechanism for permanent removal of N from aquatic ecosystem through returning of N in water to the atmosphere in the form of N₂O and N₂, although some alternative pathway, such as anaerobic ammonium oxidation (Anammox), has been discovered [5]. Denitrification in aquatic ecosystems has been widely found removing large proportion of the total N inputs to watersheds and thus providing a valuable ecosystem service by alleviating the impact of increased human N inputs [1]. Several studies have reported that the function of denitrifying bacteria communities was correlated with their abundance and community structure [6–8]. Hence, studying the variation in abundance and structure of denitrifying community will help to understand the variable denitrification potential as well as variable proportion of aquatic N removal that occurred among different aquatic ecosystems.

Streams are an important sink for anthropogenic N owing to their hydrological connections with terrestrial systems. The microbial communities in streams adapted to changes in the concentration and makeup of organic matter [9] and nutrients [10, 11]. Some recent studies have been concentrated on the link between the freshwater bacterioplankton dynamics and the environmental changes [12]. Numerous studies have reported that denitrifying bacteria can be affected by physical and chemical parameters such as pH, temperature (T), dissolved oxygen (DO), and N forms [12, 13] in series of laboratory incubation experiments. However, it is hard to identify the factors driving the variation in abundance and community structure of denitrifying bacteria in complicated aquatic ecosystems. Nowadays, functional markers include nitrite reductase (nirK and nirS) and nitrous oxide reductase (nosZ) genes have been frequently used to analyze the diversity and abundance of denitrifying bacteria community in the processes of denitrification and their response to the changing of environmental factors [8, 14–18]. Based on the analysis of these functional markers, recent research has demonstrated that the variation in the assemblage of nirS, nirK, and nosZ populations in soil was closely related to temperature, pH, and DO [19–21]. Furthermore, the abundance of these denitrifies varied in response to different nitrogen concentrations in soil, and a differential response of denitrifies communities structure to environmental gradients has also been reported [22]. These mean that functional genes of nirS, nirK, and nosZ could be sensitive indicators when studying the response of denitrifier community to variation of environmental gradients in complicated ecosystems. So far, limited studies incorporated the phosphorus concentration into the analysis of environmental gradient resulting in denitrifies community change in freshwater ecosystems [17], despite phosphorus being a vital element influencing microbial spatial patterns [23]. Therefore, in this study, we will focus on the environmental factors that have been reported to be closely related with denitrification and the variation of denitrifier community (e.g., nitrogen forms, nitrogen concentration, pH, water temperature, and DO) and the less addressed factor such as phosphorus concentration.

Dianchi Lake is the sixth largest freshwater lake in China. There are 35 streams radially flowing into the Dianchi Lake, which is a shallow plateau freshwater lake in the south-west of Kunming city, Yunnan province of China. Streams around the lake serve as ecohydrological channels that impose anthropogenic stress on the lake ecosystem and eventually cause water quality deterioration [24]. Due to the sedimentation, land reclamation, and excessive pollution, the water quality of 35 streams continued degrading from the level of drinking water quality in 1975 to the level of landscape-use only water in 2009 [24, 25]. The water quality in the north part of Dianchi Lake is the worst grade of national water quality standard. There were relatively higher and concentration even up to 12–20 mg/L in some rivers. The pH values of all sites were alkalescent [24, 26]. More than 6 sewage treatment plants (STPs) had been in operation near 11 streams in the north lake side in recent years. The effluents from the STPs are a major cause of degraded water quality in the down streams within the basin. Generally, effluents are characterized by high concentrations of nitrogen and organic matter [27]. Along with high concentration of nitrogen, many microorganisms especially denitrifying bacterial community entrained in effluents were domesticated [28].

In the present study, in order to address the response of spatial patterns of denitrifying communities to variation in environmental factors among different streams, we investigated the water properties and the abundance and diversity of denitrifying bacterial community in 11 stream inlets with different pollution sources, some of which were receiving effluents from different sewage treatment plants in the north part of Dianchi Lake, an eutrophic lake located in Southwest China (Table 1). We hypothesized that the variation in pollution sources and effluents types, such as the main eutrophication elements of N and P, may modulate the communities of denitrifiers, which may further lead to changes in the biogeochemical cycling of N in the streams and lake. The results would also provide valuable information on how the abundance and diversities of denitrifiers in various streams responded to the variation of water properties such as nitrogen forms, nitrogen concentration, phosphorus concentration, pH, water temperature, and DO. It was also expected to shed some lights on understanding the variable denitrification potential as well as variable proportion of aquatic N removal that occurred among different streams.

2. Materials and Methods

2.1. Site Description, Sampling, and Water Properties

There are 11 streams around Dianchi Lake located from 24°9′ to 25°0′ latitude and 102°6′ to 102°7′ longitude that were investigated (Figure 1).

Dissolved oxygen (DO), oxidation reduction potential (ORP), pH, and water temperature were measured in situ using portable meter (YSI ProPlus, USA) at all sampling sites. Three replicates of surface water (0–0.5 m) samples were randomly collected at three sampling locations from each sampling site of eleven streams using a cylinder sampler on 25 September 2012. Basically, the three sampling locations at each sampling site were from the upper, middle, and lower sections of a stream. One-liter water samples were reserved at −4°C with addition of Hgcl2/acid solution for chemical analysis, and two-liter water samples were filtered immediately for further molecular DNA extraction after being transported to laboratory. The concentrations of total nitrogen (TN), total phosphorus (TP), nitrate (), and phosphates () in the water samples were analyzed using a SEAL AutoAnalyzer 3 (SEAL Analytical Co., Hampshire, UK).

2.2. DNA Extraction

All water samples were kept in an ice box, transferred to the laboratory, and filtered through a 5 μm pore size sterilized filter to remove the impurities. The resultant filtrate of each sample (500 mL) was filtered through 0.22 μm Millipore membrane filters using a vacuum air pump and the membranes were stored at −80°C for DNA extraction. The membranes were cut into pieces with sterilized scissors and used immediately for DNA extraction. DNA extractions were performed using an E.Z.N.A. Water DNA Kit (OMEGA Bio-Tek Inc., Doraville, GA, USA) by following the manufacturer’s instructions. The DNA samples were stored in a −20°C freezer until use.

2.3. Real-Time Polymerase Chain Reaction Assay

The plasmids containing nirK, nirS, and nosZ fragments from environmental samples were used to create standard curve. The PCR amplified products were cloned into vector pMD19-T using the pMD19-T vector system I kit according to the manufacturer’s instructions (Takara, Dalian, China). The recombinant plasmids were inoculated into LB broth with ampicillin and incubated at 37°C overnight. Plasmid DNA was then extracted from the correct insert clones of each target gene using the E.Z.N.A. Plasmid Mini Kit II (OMEGA Bio-Tek Inc., Doraville, GA, USA) according to the manufacturer’s instructions. The plasmids DNA concentration was determined by NanoVue spectrophotometer (GE Healthcare Europe, Munich, Germany), and then the copy numbers of target genes were calculated. Tenfold serial dilutions of a known copy number of the plasmid DNA were subjected to real-time PCR assay in triplicate to generate an external standard curve.

The real-time polymerase chain reaction (qPCR) was performed on ABI 7500 real-time system (Life technologies, USA) to assess gene abundance. Amplification was performed in a 20-μL reaction mixture using SYBR Premix Ex Taq as PCR Kit provided by the suppliers (Takara bio, Dalian, China). The DNA diluted template corresponding to 1–10 ng of total DNA extracts was used in each reaction mixture. The primers and procedures used to amplify each target gene when performing real-time PCR were listed in Table 2. Data was analyzed using the 7500 software (version 2.0.6, Life technologies, USA). The parameter Ct (threshold cycle) was determined as the cycle number at which a statistically significant increase in the reporter fluorescence was detected. Standard curves for real-time PCR assays were created according to the method described by Henry et al. [34].

2.4. PCR Amplification Denaturing Gradient Gel Electrophoresis Analysis

The community structures of nirK, nirS, and nosZ genes were analyzed by denaturing gradient gel electrophoresis (DGGE). The amplification was performed in 50-μL reaction mixtures including 1x PCR buffer, 400 μmol/L of each dNTP, and 2.5 U hot star Taq DNA polymerase (Takara Bio, Otsu, Shiga, Japan) plus primers (Table 2).

The amplified products were pooled and resolved on DGGE gels using a Dcode system (Bio-Rad Laboratories Inc. Hercules, USA). PCR samples (50 μL) containing approximately equal amounts of PCR amplicons were loaded onto the 1 mm thick 8% (w/v) polyacrylamide (37.5 : 1, acrylamide : bisacrylamide) gels in 1x TAE buffer (40 mM Tris-acetate and 1 mM EDTA) with denaturing gradients of 50–75% for 15 h (nirS), 50–70% for 12 h (nirK), and 50–70% (nosZ) (100% denaturant contains 7 mol/L urea and 40% (v/v) formamide) for 15 h at 100 V and 60°C, respectively. After being stained with silver nitrate according to the protocol [35], polaroid pictures of the DGGE gels were scanned using an EPSON (Perfection V700 Photo) scanner and stored as TIFF files and digitized and then analyzed with the Quantity One software (version 4.5, Bio-Rad, USA).

2.5. Data Analysis

Three replicates were used in all parameter analysis. Data were presented as the mean values of triplicates and the maximum difference (mean ± SD) among triplicate results was 5%. One way analysis of variance (ANOVA) was performed to test whether there were any significant differences among the means at the 95% confidence level. Potential relationships between all denitrifying bacteria abundance and environmental data sets were tested by Pearson correlation analysis. All data were analysed using SPSS software.

DGGE banding profiles for nirS, nirK, and nosZ communities were digitized after average background subtraction for entire gels. Band position and intensity date for each sample were exported to an excel spreadsheet prior to further statistical analyses. The relative intensity of a specific band was transformed according to the sum of intensities of all bands in a pattern [36]. Redundancy analysis (RDA) for community ordination was conducted using CANOCO (version 4.5, Centre for Biometry, Wageningen, The Netherlands) for Windows using relative band intensity data obtained from the Quantity One analysis [37, 38]. Among all environmental variables, eight parameters, including water temperature, pH, DO, oxidation reduction potential nitrate, total nitrogen, total phosphorus (TP), and phosphates (), were selected to perform RDA by Monte Carlo reduced model tests with 499 unrestricted permutations to statistically evaluate the significance of the first canonical axis and of all canonical axes together. Statistical significance was kept at for all analyses (Table 4).

3. Results

3.1. Spatial Patterns of Structure and Size of Denitrifier Communities

All digitized data of the three replicates were used in statistics analysis. The Shannon index () of nirK, nirS, and nosZ calculated from DGGE gels ranged from 2.08 to 2.64, 1.73 to 2.80, and 1.89 to 2.60, respectively. The significant differences among them were observed statistically (). There were relative lower richness and diversity of nirK, nirS, and nosZ in Xinyunliang stream (Table 3), while the highest of the Shannon values and richness of nirK, nirS, and nosZ occurred in Guangpugou stream.

The results of qPCR showed that the abundance of nirS, nirK, and nosZ gene copies per mL water ranged from 2.79 × 10³ to 1.20 × 10⁵, 3.23 × 10³ to 1.76 × 10⁵, and 8.66 × 10² to 1.90 × 10⁵, respectively. The abundance of nirK, nirS, and nosZ denitrifiers in Xinbaoxiang, Daguan, Chuanfang, and Panlongjiang streams was relatively stable and low. The results of nirK, nirS, and nosZ genes abundance in group Xiaba and Yaoan streams were consistent. The absolute abundance of denitrification genes in the other five streams varied widely (Figure 2). These results indicated that different pollution source would influence the abundance of denitrifiers in streams. The denitrifiers (nirK, nirS, and nosZ) abundance of the water samples in Haihe stream and Guangpugou stream was significantly higher than that in other stream samples (). The abundance of nirK- and nosZ-type denitrifiers had similar trends in the streams, with higher number in Haihe stream and Guangpugou stream and intermediate levels in Xinbaoxiang, Xiaba, Yaoan, Jinjia, Panlongjiang, and Xiaba streams but lower levels in Chuangfang stream, Daguang stream, and Xinyunliang stream. However, the nirS-type abundance in all sites did not totally follow this trend and no significant differences were detected in qPCR data among Haihe, Guangpugou, and Daguang streams.

Ratios of nosZ abundance to the abundance of nirK + nirS for all samples varied widely. The highest nosZ/(nirK + nirS) ratio occurred in Haihe stream (0.64), the lowest nosZ/(nirK + nirS) ratio occurred in Daguang stream (0.08), and the nosZ/(nirK + nirS) ratios in the other streams were similar and ranged from 0.13 to 0.35. Ratios of nirK abundance nirK/nirS to nirS abundance in different streams were also different. The highest (nirK/nirS) ratio occurred in Daguang stream (18.79), the lowest (nirK/nirS) ratios occurred in Xinyunliang stream (0.48), and the (nirK/nirS) ratios in the other streams were between 0.90 and 2.15. These results implied that the abundance of nirS was not always greater than that of nirK in stream inlet water column.

3.2. Water Parameters Controlling Denitrifier Communities

In order to determine to what extent the eight environmental properties affected the three types of denitrifying genes on their community compositions, DGGE fingerprints were analyzed by redundancy analysis. The results showed that , pH, and water temperature were the relatively important environmental parameters for denitrifiers (Table 5). For nirK-type denitrifier, , TN, and pH explained 50% variations of microbial communities. Variation partitioning analysis showed that , TN, and pH separately explained 21% (), 19% (), and 10% () of the variation, respectively. The analysis did not reveal significant relationship between nirS-type denitrifier communities and any environmental parameters. For nosZ-type denitrifier, temperature (17%, ), (13%, ), and total (8%, ) explained 38% variations of microbial communities.

nirS abundance was significantly and positively correlated with nirK and nosZ abundance. nirK abundance was significantly correlated in a positive direction with nosZ abundance. These results suggested that all of three denitrifiers can interact with each other. The abundance diversification of nirK, nirS, and nosZ was strongly and positively associated with TN and (). The analysis did not reveal significant relationship between pH and any denitrifying bacteria gene abundance. All relationships between nirS, nirK, and nosZ genes abundance and chemical variables were positively correlated except for DO, ORP, T, and , which were negatively correlated with the copy numbers of nirS, nirK, and nosZ genes. The concentration was a key parameter influencing the ratios of nirK abundance of nirS. A significant correlated correlation existed between ratios of nosZ abundance to the abundance of nirK + nirS and the concentration total nitrogen.

4. Discussion

4.1. The Variation of Community Pattern of Denitrifying Bacteria according to Pollution Sources and Effluent Types of Different Streams

Nitrogen cycle in aquatic ecosystems is predominantly controlled by bacteria, and their activities determine the fate of nitrogen compounds. Meanwhile, environmental conditions that regulate the activity of bacteria determine where each nitrogen transformation process occurs and the degree of exchange among various nitrogen pools. Thus, chemical information of different nitrogen species alone is not sufficient to predict rates of nitrogen transformation processes in the environment, and information concerning characteristics of nitrogen cycling bacterial community under various environmental conditions is essential for understanding the related nitrogen cycle process. With regard to the denitrification process, previous studies have shown that it was regulated by various environmental factors such as oxygen and nitrogen concentration, quality, temperature, and pH [5, 39]. However, how the denitrifying bacteria communities were correlated with the environmental factors in streams, receiving massive amount of nutrients and pollutants, remains unclear.

In this study, we evaluated differences in the genetic makeup of the communities by comparing DGGE profiles for denitrification genes encoding nitrite and nitrous oxide reductase (nirK, nirS, and nosZ). It has been suggested that DGGE was a powerful tool for identifying and comparing the dominant of these communities [40, 41]. The DGGE results in this study revealed the significant pairwise differences in the community structure of denitrifying bacteria containing the nirK, nirS, or nosZ genes among the water samples collected from different streams. When comparing the diversities of denitrifier communities from all sites, similar trends emerged with low richness and diversity in the Xinyunliang stream. According to previous studies, Xinyunliang streams run through a historical area of old Kunming city, where there are many industries (such as Yunnan smelter), high population density, and poor sewage networks [42]. This serious industrial pollution could reduce bacterial diversity and damage microbial ecological system [43, 44], although the concentration of , the substrates for denitrification, in Xinyunliang stream was higher than most of other streams with exception of Guangpugou and Haihe streams. Our studied streams differed substantially in the amount of inorganic nutrients which were potentially available to denitrifiers and other microbial populations during the development of microbial community in the water column. The three large streams around Dianchi Lake, Panlongjiang, Daguan, and Chuanfang streams [42], were the important sites receiving effluents from the STPs, with the characteristics of high nitrate concentrations. Nevertheless, the fast-flowing water and irregular discharge of effluents prevented the stream from developing a stable environment for microbial colonization and propagation. Therefore, these streams also represented relatively low diversities of denitrifying bacteria. It has been reported that the high stream flow and nitrate concentration of streams were the major factors controlling the development of planktonic denitrifier populations [45]. On the contrary, the streams of Haihe and Guangpugou were of narrow and slow-flowing, which leading to the long residence time for nitrogen-containing pollutants and well-established hypoxic (~0.2 mg L⁻¹ in DO) environment. Therefore, the abundance of denitrifiers was much higher in Haihe and Guangpugou streams than the other streams, which may enhance denitrification in Haihe and Guangpugou streams [46]. This result was consistent with the previous reports that the most transformation of inorganic nitrogen occurred in narrow streams [47]. Simultaneously, an ecological engineering project using Eichhornia crassipes for nutrient removal has been conducted in the 11 streams around Dianchi Lake since June 2011. The roots of E. crassipes [48, 49] in streams provided a large specific area for denitrifiers to attach, which would benefit the formation of biofilms and therefore may further change or modify the diversity and abundance of denitrifiers in water [50]. Previous studies suggested that microbial biofilms were highly efficient and successful ecological communities that might also contribute to the influence of the headwater streams on streams, estuaries, and even oceans [51]. Therefore, the slow-moving flows such as Haihe and Guangpugou streams could be considered as living zones of transient storage, where roots and other biofilms bring hydrodynamic retention and biochemical processing into close spatial proximity and influence biogeochemical processes and patterns in streams. All of these results coincided with our hypothesis that the pollution sources and effluent types of different streams would modulate the community composition of denitrifying bacteria to a great extent, although a complex picture of denitrifier community similarity emerged depending on which functional denitrification gene was evaluated.

In this study, we found that the distribution of nir-denitrification genes was much patchier, which was consistent with earlier observations in other streams [52, 53]. Studies of changes in composition and diversity of nirK and nirS genes communities support the hypothesis of niche differentiation among denitrifying bacteria [54–56]. Meanwhile, this study found the abundance of nirS was not consistently greater than that of nirK in stream inlet water column, which was different from some of the previously published results [53, 57]. However, in a similar way, some of previous studies also suggested that the spatial distribution of nirS and nirK genes abundance differs in other types of urban streams, reflecting different habitat preferences [52, 53]. The results of the nirK/nirS ratios suggested that the nirK-type denitrifiers might be more abundant than nirS-type denitrifiers in Daguang stream (nirK/nirS ratio, 18.79), in contrast to Panlongjiang stream (nirK/nirS ratio, 0.90). However, the concentrations of DO in the two streams were both relatively higher (~2.8 mg L⁻¹) than others. This contradicted with previous studies that nirK often prevailed in conditionally O₂-exposed environments [53, 58]. This discrepancy probably was due to other environmental parameters such as nitrogen and phosphorus concentrations [21, 53] which varied significantly in the two streams. Even though the nirK and nirS are functionally equivalent, denitrifying bacteria harboring either nitrite reductase was likely not under the same community assembly rules [59]. Philippot et al. [60] suggested that the existence of the two types of nitrite reductase (nir-gene) was due to differential niche preferences. The different community patterns, together with the spatial distribution of the nirS/nirK abundance ratio, can suggest habitat selection for the nirS- and nirK-type denitrifiers [17]. In the present study, the different spatial patterns of nirK and nirS community diversities, in combination with the spatial distribution of the nirS/nirK ratio, indicated habitat selection for the two types of denitrifiers. Denitrifying organism includes either nirS or nirK, but not both of the two-type nitrite reductase genes [61, 62], and experiments have shown the two nitrite reductases to be functionally redundant, as one nir-type gene in denitrifying bacteria can be eliminated and replaced by the other type [63]. This, however, did not necessarily indicate that nirK-type denitrifiers contributed more or less in denitrification than nirK-type ones. Hence, gene expression analysis is necessary to further investigate which is more important in denitrification in the stream inlet water column around Dianchi Lake.

4.2. Relationship between Water Properties and Spatial Patterns of Denitrifying Bacteria

In the present study, the RDA charts of nirK, nirS, and nosZ genes indicated that diversity of denitrifying populations had varying response to environmental factors, and the concentrations of P ( or TP) and N ( or TN) were the most important environmental factors causing a significant alteration in the denitrifier community structure among different streams by serving as essential nutrients for microorganisms growth in streams. Meanwhile, abundance of all denitrifiers in this study was, by and large, controlled by the water parameters, especially nutrient (phosphorous and nitrogen) concentration (Table 3). Certainly, microbes need phosphorus for their growth and function. Finlay et al. found increasing phosphorous inputs associated with eutrophication can indirectly promote N losses via enhancing denitrification [64]. Additions of P have been demonstrated to increase N removal in whole-ecosystem experiments in both lakes and streams [65], which provides further support for the role of P as an important control over N cycling and fate in freshwater ecosystems. In addition, different responses of community diversities of nirS- and nirK-denitrifiers to the changes of phosphorous concentrations agreed with a study suggesting that nirK-denitrifiers were most sensitive to alteration of phosphorous concentration [21]. Contrary to previous studies [17, 46], our results implied that variation of phosphorous content in water was positively linked to the abundance of nirS and nirK genes and resulted in shift of community structure of nir-containing denitrifiers populations. This may further clarify the important function of phosphorous in shaping microorganisms structure in environments [23, 46]. However, the mechanisms concerning how phosphorus would affect growth of denitrifying bacteria in water are still not well understood [21]. Further studies are needed to explain underlying mechanism related to the role of P in regulating the denitrifiers’ community, although our results have built some supporting evidence for the related phenomenon. In addition, the water temperature and pH were the main factors driving the changes in the denitrifying bacterial community composition among different streams. The genes nirS, nirK, and nosZ abundance was shown by Pearson correlation coefficient () to be mightily influenced by water temperature and oxidation reduction potential (ORP). The quality of inlet water in 11 streams differed with water origin and pollution sources [24, 25, 42]. It has been shown that the changes in denitrifying community structures responded to their habitat conditions like temperature and DO gradient and N forms [12, 13, 66, 67]. The pH was known to generally affect denitrifier community diversity and richness [15]. Generally, the effect of temperature on driving biogeochemical processes is either to alter the functioning bacteria without changing the microbial communities or restructuring communities, thus modifying the fundamental physiologies [68]. Previous studies suggested that temperature could directly or indirectly affect the communities’ diversity and abundance of denitrifying organisms [16, 20, 69–71].

5. Conclusions

The results showed that abundance and diversities of denitrifying genes (nirK, nirS, and nosZ) were variable in the streams of Dianchi Lake. Nutrient concentrations (nitrogen and phosphorous), water temperature, and pH were important environmental factors to alter abundance and community structure of the denitrifiers significantly. The different community patterns, together with the spatial distribution of the nirS/nirK abundance ratio, suggest habitat selection for the nirS- and nirK-type denitrifiers.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Neng Yi and Yan Gao contributed equally to this paper.

Acknowledgments

The authors are grateful for the financial support from State Natural Science Foundation of China (no. 41401592), Natural Science Foundation of Jiangsu Province (no. BK20140737), Jiangsu Province Science and Technology Support Plan Project (BE2013436), and Independent Innovation of Agricultural Sciences in Jiangsu Province (no. CX(14)5047).

References

P. J. Mulholland, A. M. Helton, G. C. Poole et al., “Stream denitrification across biomes and its response to anthropogenic nitrate loading,” Nature, vol. 452, no. 7184, pp. 202–205, 2008.
View at: Publisher Site | Google Scholar
S. Seitzinger, J. A. Harrison, J. K. Böhlke et al., “Denitrification across landscapes and waterscapes: a synthesis,” Ecological Applications, vol. 16, no. 6, pp. 2064–2090, 2006.
View at: Publisher Site | Google Scholar
F. Azam and A. Z. Worden, “Microbes, molecules, and marine ecosystems,” Science, vol. 303, no. 5664, pp. 1622–1624, 2004.
View at: Publisher Site | Google Scholar
K. Song, H. Kang, L. Zhang, and W. J. Mitsch, “Seasonal and spatial variations of denitrification and denitrifying bacterial community structure in created riverine wetlands,” Ecological Engineering, vol. 38, no. 1, pp. 130–134, 2012.
View at: Publisher Site | Google Scholar
J. Vymazal, “Removal of nutrients in various types of constructed wetlands,” Science of the Total Environment, vol. 380, no. 1–3, pp. 48–65, 2007.
View at: Publisher Site | Google Scholar
D. Chèneby, A. Hartmann, C. Hénault, E. Topp, and J. C. Germon, “Diversity of denitrifying microflora and ability to reduce N₂O in two soils,” Biology and Fertility of Soils, vol. 28, no. 1, pp. 19–26, 1998.
View at: Publisher Site | Google Scholar
E. Kandeler, T. Brune, E. Enowashu et al., “Response of total and nitrate-dissimilating bacteria to reduced N deposition in a spruce forest soil profile,” FEMS Microbiology Ecology, vol. 67, no. 3, pp. 444–454, 2009.
View at: Publisher Site | Google Scholar
S. E. Morales, T. Cosart, and W. E. Holben, “Bacterial gene abundances as indicators of greenhouse gas emission in soils,” The ISME Journal, vol. 4, no. 6, pp. 799–808, 2010.
View at: Publisher Site | Google Scholar
D. L. Kirchman, A. I. Dittel, S. E. G. Findlay, and D. Fischer, “Changes in bacterial activity and community structure in response to dissolved organic matter in the Hudson River, New York,” Aquatic Microbial Ecology, vol. 35, no. 3, pp. 243–257, 2004.
View at: Publisher Site | Google Scholar
A. Cébron, T. Berthe, and J. Garnier, “Nitrification and nitrifying bacteria in the lower Seine River and estuary (France),” Applied and Environmental Microbiology, vol. 69, no. 12, pp. 7091–7100, 2003.
View at: Publisher Site | Google Scholar
A. Cébron, M. Coci, J. Garnier, and H. J. Laanbroek, “Denaturing gradient gel electrophoretic analysis of ammonia-oxidizing bacterial community structure in the lower seine river: impact of paris wastewater effluents,” Applied and Environmental Microbiology, vol. 70, no. 11, pp. 6726–6737, 2004.
View at: Publisher Site | Google Scholar
Z. Liu, S. Huang, G. Sun, Z. Xu, and M. Xu, “Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China,” FEMS Microbiology Ecology, vol. 80, no. 1, pp. 30–44, 2012.
View at: Publisher Site | Google Scholar
B. C. Crump and J. E. Hobbie, “Synchrony and seasonality in bacterioplankton communities of two temperate rivers,” Limnology and Oceanography, vol. 50, no. 6, pp. 1718–1729, 2005.
View at: Publisher Site | Google Scholar
K. Enwall, L. Philippot, and S. Hallin, “Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization,” Applied and Environmental Microbiology, vol. 71, no. 12, pp. 8335–8343, 2005.
View at: Publisher Site | Google Scholar
S. Hallin, C. M. Jones, M. Schloter, and L. Philippot, “Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment,” The ISME Journal, vol. 3, no. 5, pp. 597–605, 2009.
View at: Publisher Site | Google Scholar
M. Wolsing and A. Priemé, “Observation of high seasonal variation in community structure of denitrifying bacteria in arable soil receiving artificial fertilizer and cattle manure by determining T-RFLP of nir gene fragments,” FEMS Microbiology Ecology, vol. 48, no. 2, pp. 261–271, 2004.
View at: Publisher Site | Google Scholar
K. Enwall, I. N. Throbäck, M. Stenberg, M. Söderström, and S. Hallin, “Soil resources influence spatial patterns of denitrifying communities at scales compatible with land management,” Applied and Environmental Microbiology, vol. 76, no. 7, pp. 2243–2250, 2010.
View at: Publisher Site | Google Scholar
L. Philippot, J. Andert, C. M. Jones, D. Bru, and S. Hallin, “Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N₂O emissions from soil,” Global Change Biology, vol. 17, no. 3, pp. 1497–1504, 2011.
View at: Publisher Site | Google Scholar
J. Čuhel, M. Šimek, R. J. Laughlin et al., “Insights into the effect of soil pH on N₂O and N₂ emissions and denitrifier community size and activity,” Applied and Environmental Microbiology, vol. 76, no. 6, pp. 1870–1878, 2010.
View at: Publisher Site | Google Scholar
J. K. M. Walker, K. N. Egger, and G. H. R. Henry, “Long-term experimental warming alters nitrogen-cycling communities but site factors remain the primary drivers of community structure in high arctic tundra soils,” The ISME Journal, vol. 2, no. 9, pp. 982–995, 2008.
View at: Publisher Site | Google Scholar
T. Yan, M. W. Fields, L. Wu, Y. Zu, J. M. Tiedje, and J. Zhou, “Molecular diversity and characterization of nitrite reductase gene fragments (nirK and nirS) from nitrate-and uranium-contaminated groundwater,” Environmental Microbiology, vol. 5, no. 1, pp. 13–24, 2003.
View at: Publisher Site | Google Scholar
K. Hamonts, T. J. Clough, A. Stewart et al., “Effect of nitrogen and waterlogging on denitrifier gene abundance, community structure and activity in the rhizosphere of wheat,” FEMS Microbiology Ecology, vol. 83, no. 3, pp. 568–584, 2013.
View at: Publisher Site | Google Scholar
J.-Z. He, Y. Zheng, C.-R. Chen, Y.-Q. He, and L.-M. Zhang, “Microbial composition and diversity of an upland red soil under long-term fertilization treatments as revealed by culture-dependent and culture-independent approaches,” Journal of Soils and Sediments, vol. 8, no. 5, pp. 349–358, 2008.
View at: Publisher Site | Google Scholar
Y. Huang, H. Wen, J. Cai, M. Cai, and J. Sun, “Key aquatic environmental factors affecting ecosystem health of streams in the Dianchi Lake Watershed, China,” Procedia Environmental Sciences, vol. 2, no. 10, pp. 868–880, 2010.
View at: Publisher Site | Google Scholar
Y. J. Yu, J. Guan, Y. W. Ma, S. Yu, H. Guo, and L. Bao, “Aquatic environmental quality variation in Lake Dianchi watershed,” Procedia Environmental Sciences, vol. 2, no. 10, pp. 76–81, 2010.
View at: Publisher Site | Google Scholar
State Environmental Protection Administration, The National Standard of The People's Republic of China-Surface Water Environmental Quality Standards, China Environmental Science Press, 2002.
Ncdenr, Basin Wide Assessment Report: Cape Fear River Basin, Division of Water Quality, Department of Environment and Natural Resources, Raleigh, NC, USA, 2004.
C. Féray and B. Montuelle, “Chemical and microbial hypotheses explaining the effect of wastewater treatment plant discharges on the nitrifying communities in freshwater sediment,” Chemosphere, vol. 50, no. 7, pp. 919–928, 2003.
View at: Publisher Site | Google Scholar
K. Kloos, A. Mergel, C. Rösch, and H. Bothe, “Denitrification within the genus azospirillum and other associative bacteria,” Australian Journal of Plant Physiology, vol. 28, no. 9, pp. 991–998, 2001.
View at: Google Scholar
I. N. Throbäck, K. Enwall, Å. Jarvis, and S. Hallin, “Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE,” FEMS Microbiology Ecology, vol. 49, no. 3, pp. 401–417, 2004.
View at: Publisher Site | Google Scholar
V. Michotey, V. Méjean, and P. Bonin, “Comparison of methods for quantification of cytochrome cd₁-denitrifying bacteria in environmental marine samples,” Applied and Environmental Microbiology, vol. 66, no. 4, pp. 1564–1571, 2000.
View at: Publisher Site | Google Scholar
G. Braker and J. M. Tiedje, “Nitric oxide reductase (norB) genes from pure cultures and environmental samples,” Applied and Environmental Microbiology, vol. 69, no. 6, pp. 3476–3483, 2003.
View at: Publisher Site | Google Scholar
S. Hallin and P.-E. Lindgren, “PCR detection of genes encoding nitrite reductase in denitrifying bacteria,” Applied and Environmental Microbiology, vol. 65, no. 4, pp. 1652–1657, 1999.
View at: Google Scholar
S. Henry, D. Bru, B. Stres, S. Hallet, and L. Philippot, “Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils,” Applied and Environmental Microbiology, vol. 72, no. 8, pp. 5181–5189, 2006.
View at: Publisher Site | Google Scholar
D. Radojkovic and J. Kusic, “Silver staining of denaturing gradient gel electrophoresis gels,” Clinical Chemistry, vol. 46, no. 6, pp. 883–884, 2000.
View at: Google Scholar
D. Roesti, R. Gaur, B. N. Johri et al., “Plant growth stage, fertiliser management and bio-inoculation of arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria affect the rhizobacterial community structure in rain-fed wheat fields,” Soil Biology and Biochemistry, vol. 38, no. 5, pp. 1111–1120, 2006.
View at: Publisher Site | Google Scholar
J. Lepš and P. Šmilauer, Multivariate Analysis of Ecological Data using CANOCO, Cambridge University Press, Cambridge, UK, 2003.
View at: Publisher Site
J. Zhang, G. Zeng, Y. Chen et al., “Effects of physico-chemical parameters on the bacterial and fungal communities during agricultural waste composting,” Bioresource Technology, vol. 102, no. 3, pp. 2950–2956, 2011.
View at: Publisher Site | Google Scholar
J. L. Faulwetter, V. Gagnon, C. Sundberg et al., “Microbial processes influencing performance of treatment wetlands: a review,” Ecological Engineering, vol. 35, no. 6, pp. 987–1004, 2009.
View at: Publisher Site | Google Scholar
Z.-F. Zhou, Y.-M. Zheng, J.-P. Shen, L.-M. Zhang, and J.-Z. He, “Response of denitrification genes nirS, nirK, and nosZ to irrigation water quality in a Chinese agricultural soil,” Environmental Science and Pollution Research, vol. 18, no. 9, pp. 1644–1652, 2011.
View at: Publisher Site | Google Scholar
Z. Jin, F.-Y. Ji, X. Xu, X.-Y. Xu, Q.-K. Chen, and Q. Li, “Microbial and metabolic characterization of a denitrifying phosphorus-uptake/side stream phosphorus removal system for treating domestic sewage,” Biodegradation, vol. 25, no. 6, pp. 777–786, 2014.
View at: Publisher Site | Google Scholar
B. Wang, B. Huang, W. Jin et al., “Occurrence, distribution, and sources of six phenolic endocrine disrupting chemicals in the 22 river estuaries around Dianchi Lake in China,” Environmental Science and Pollution Research, vol. 20, no. 5, pp. 3185–3194, 2013.
View at: Publisher Site | Google Scholar
J. Xiong, Z. He, J. D. Van Nostrand et al., “Assessing the microbial community and functional genes in a vertical soil profile with long-term arsenic contamination,” PLoS ONE, vol. 7, no. 11, Article ID e50507, 2012.
View at: Publisher Site | Google Scholar
H. M. A. Mahmoud, R. Goulder, and G. R. Carvalho, “The response of epilithic bacteria to different metals regime in two upland streams: assessed by conventional microbiological methods and PCR-DGGE,” Archiv für Hydrobiologie, vol. 163, no. 3, pp. 405–427, 2005.
View at: Publisher Site | Google Scholar
J. K. Böhlke, R. C. Antweiler, J. W. Harvey et al., “Multi-scale measurements and modeling of denitrification in streams with varying flow and nitrate concentration in the upper Mississippi River basin, USA,” Biogeochemistry, vol. 93, no. 1-2, pp. 117–141, 2009.
View at: Publisher Site | Google Scholar
C. Yin, F. L. Fan, A. Song, Z. Li, W. Yu, and Y. Liang, “Different denitrification potential of aquic brown soil in Northeast China under inorganic and organic fertilization accompanied by distinct changes of nirS- and nirK-denitrifying bacterial community,” European Journal of Soil Biology, vol. 65, pp. 47–56, 2014.
View at: Publisher Site | Google Scholar
B. J. Peterson, W. M. Wollheim, P. J. Mulholland et al., “Control of nitrogen export from watersheds by headwater streams,” Science, vol. 292, no. 5514, pp. 86–90, 2001.
View at: Publisher Site | Google Scholar
Y. Gao, N. Yi, Z. Zhang, H. Liu, and S. Yan, “Fate of ¹⁵NO₃⁻ and ¹⁵NH₄⁺ in the treatment of eutrophic water using the floating macrophyte, Eichhornia crassipes,” Journal of Environmental Quality, vol. 41, no. 5, pp. 1653–1660, 2012.
View at: Publisher Site | Google Scholar
N. Yi, Y. Gao, X.-H. Long et al., “Eichhornia crassipes cleans wetlands by enhancing the nitrogen removal and modulating denitrifying bacteria community,” CLEAN—Soil, Air, Water, vol. 42, no. 5, pp. 664–673, 2014.
View at: Publisher Site | Google Scholar
B. Wei, X. Yu, S. Zhang, and L. Gu, “Comparison of the community structures of ammonia-oxidizing bacteria and archaea in rhizoplanes of floating aquatic macrophytes,” Microbiological Research, vol. 166, no. 6, pp. 468–474, 2011.
View at: Publisher Site | Google Scholar
T. J. Battin, L. A. Kaplan, J. D. Newbold, and C. M. E. Hansen, “Contributions of microbial biofilms to ecosystem processes in stream mesocosms,” Nature, vol. 426, no. 6965, pp. 439–442, 2003.
View at: Publisher Site | Google Scholar
P. M. Groffman, A. M. Dorsey, and P. M. Mayer, “N processing within geomorphic structures in urban streams,” Journal of the North American Benthological Society, vol. 24, no. 3, pp. 613–625, 2005.
View at: Publisher Site | Google Scholar
C. W. Knapp, W. K. Dodds, K. C. Wilson, J. M. O'Brien, and D. W. Graham, “Spatial heterogeneity of denitrification genes in a highly homogenous urban stream,” Environmental Science & Technology, vol. 43, no. 12, pp. 4273–4279, 2009.
View at: Publisher Site | Google Scholar
S. Hallin, I. N. Throbäck, J. Dicksved, and M. Pell, “Metabolic profiles and genetic diversity of denitrifying communities in activated sludge after addition of methanol or ethanol,” Applied and Environmental Microbiology, vol. 72, no. 8, pp. 5445–5452, 2006.
View at: Publisher Site | Google Scholar
B. B. Oakley, C. A. Francis, K. J. Roberts, C. A. Fuchsman, S. Srinivasan, and J. T. Staley, “Analysis of nitrite reductase (nirK and nirS) genes and cultivation reveal depauperate community of denitrifying bacteria in the Black Sea suboxic zone,” Environmental Microbiology, vol. 9, no. 1, pp. 118–130, 2007.
View at: Publisher Site | Google Scholar
J. M. Smith and A. Ogram, “Genetic and functional variation in denitrifier populations along a short-term restoration chronosequence,” Applied and Environmental Microbiology, vol. 74, no. 18, pp. 5615–5620, 2008.
View at: Publisher Site | Google Scholar
A. García-Lledó, A. Vilar-Sanz, R. Trias, S. Hallin, and L. Bañeras, “Genetic potential for N₂O emissions from the sediment of a free water surface constructed wetland,” Water Research, vol. 45, no. 17, pp. 5621–5632, 2011.
View at: Publisher Site | Google Scholar
D. W. Graham, C. Trippett, W. K. Dodds et al., “Correlations between in situ denitrification activity and nir-gene abundances in pristine and impacted prairie streams,” Environmental Pollution, vol. 158, no. 10, pp. 3225–3229, 2010.
View at: Publisher Site | Google Scholar
C. M. Jones and S. Hallin, “Ecological and evolutionary factors underlying global and local assembly of denitrifier communities,” The ISME Journal, vol. 4, no. 5, pp. 633–641, 2010.
View at: Publisher Site | Google Scholar
L. Philippot, J. Čuhel, N. P. A. Saby et al., “Mapping field-scale spatial patterns of size and activity of the denitrifier community,” Environmental Microbiology, vol. 11, no. 6, pp. 1518–1526, 2009.
View at: Publisher Site | Google Scholar
C. M. Jones, B. Stres, M. Rosenquist, and S. Hallin, “Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification,” Molecular Biology and Evolution, vol. 25, no. 9, pp. 1955–1966, 2008.
View at: Publisher Site | Google Scholar
W. G. Zumft, “Cell biology and molecular basis of denitrification,” Microbiology and Molecular Biology Reviews, vol. 61, no. 4, pp. 533–616, 1997.
View at: Google Scholar
A. B. Glockner, A. Jüngst, and W. G. Zumft, “Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd₁-free background (NirS⁻-) of Pseudomonas stutzeri,” Archives of Microbiology, vol. 160, no. 1, pp. 18–26, 1993.
View at: Publisher Site | Google Scholar
J. C. Finlay, G. E. Small, and R. W. Sterner, “Human influences on nitrogen removal in lakes,” Science, vol. 342, no. 6155, pp. 247–250, 2013.
View at: Publisher Site | Google Scholar
E. S. Bernhardt, “Cleaner lakes are dirtier lakes,” Science, vol. 342, no. 6155, pp. 205–206, 2013.
View at: Publisher Site | Google Scholar
J. J. Rich, R. S. Heichen, P. J. Bottomley, K. Cromack Jr., and D. D. Myrold, “Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils,” Applied and Environmental Microbiology, vol. 69, no. 10, pp. 5974–5982, 2003.
View at: Publisher Site | Google Scholar
M. Castro-González, G. Braker, L. Farías, and O. Ulloa, “Communities of nirS-type denitrifiers in the water column of the oxygen minimum zone in the eastern South Pacific,” Environmental Microbiology, vol. 7, no. 9, pp. 1298–1306, 2005.
View at: Publisher Site | Google Scholar
J. P. Schimel and J. Gulledge, “Microbial community structure and global trace gases,” Global Change Biology, vol. 4, no. 7, pp. 745–758, 1998.
View at: Publisher Site | Google Scholar
G. Braker, J. Schwarz, and R. Conrad, “Influence of temperature on the composition and activity of denitrifying soil communities,” FEMS Microbiology Ecology, vol. 73, no. 1, pp. 134–148, 2010.
View at: Publisher Site | Google Scholar
L. Holtan-Hartwig, P. Dörsch, and L. R. Bakken, “Low temperature control of soil denitrifying communities: kinetics of N₂O production and reduction,” Soil Biology and Biochemistry, vol. 34, no. 11, pp. 1797–1806, 2002.
View at: Publisher Site | Google Scholar
L. Zhang, G. Zeng, J. Zhang et al., “Response of denitrifying genes coding for nitrite (nirK or nirS) and nitrous oxide (nosZ) reductases to different physico-chemical parameters during agricultural waste composting,” Applied Microbiology and Biotechnology, vol. 99, no. 9, pp. 4059–4070, 2015.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2015 Neng Yi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

1720

Downloads

1133

Citations