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Nitrogen loading affects microbes, nitrifiers and denitrifiers attached to submerged macrophyte in constructed wetlands
Science of the Total Environment 622–623 (2018) 121–126
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Nitrogen loading affects microbes, nitrifiers and denitrifiers attached to submerged macrophyte in constructed wetlands Liying Yan a, Songhe Zhang a,⁎, Da Lin a, Chuan Guo a, Lingling Yan a, Supeng Wang a, Zhenli He b a b
Ministry of Education Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, College of Environment, Hohai University, Nanjing 210098, China University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, Fort Pierce, FL 34945, USA
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Initial nitrogen supply stimulated biofilm growth and algae density. • Submerged plants performed better in the removal of nitrogen than artificial plants. • Nitrogen and the species of plant determined algae composition in biofilm. • Nitrogen loading stimulated the abundance of nitrifiers and denitrifiers.
a r t i c l e
i n f o
Article history: Received 24 October 2017 Received in revised form 19 November 2017 Accepted 20 November 2017 Available online xxxx
Keywords: Biofilms Denitrification Nitrification Submerged macrophyte Algae
a b s t r a c t Submerged macrophytes and biofilms are important components of wetlands. However, little is known about the changes of microbes in biofilms attached to submerged macrophytes upon nitrogen loading. This study investigated the changes of microbes, algae, nitrifiers and denitrifiers in biofilms attached to the leaves of artificial plants (AP), Potamogeton malaianus (PM), Vallisneria natans (VN) and Hydrilla verticillata (HV) under varied initial concentrations of total nitrogen (TN). Nitrogen addition increased biofilm biomass and changed dissolved oxygen concentrations and pH values in overlaying water. Epiphytic algal densities showed the same trend at the same N level:AP N PM N VN N HV. As revealed by cluster analysis at phylum level, algae compositions in biofilm from four plants showed some host-specific at 2 and 12 mg L−1 TN, but was clustered in the same group at 22 mg L−1 TN regardless of plant species. Submerged macrophytes had better performance in total N removal than AP. In general, N application significantly increased the abundance of amoA, nirK, nirS, napA and cnorB in biofilm. The abundance of the denitrification genes (nirK, nirS, napA, narG and cnorB) was positively correlated with nitrogen application, while amoA was correlated with concentration of dissolved oxygen. These results indicate that N loadings stimulated the growth of biofilms attached to submerged macrophyte and the removal of total N can be partially ascribed to the synergistic interactions of submerged macrophyte and biofilms in wetlands. These results highlight the ecological role of submerged macrophyte-biofilm system in nitrogen removal in wetlands. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Eutrophication of freshwater has become a global problem due to input of large amounts of nitrogen (N) and phosphorus (P) from ⁎ Corresponding author. E-mail address: [email protected] (S. Zhang).
https://doi.org/10.1016/j.scitotenv.2017.11.234 0048-9697/© 2017 Elsevier B.V. All rights reserved.
agriculture and urban areas (De-Bashan and Bashan, 2010). Eutrophication often causes algae bursts and subsequent decline of submerged macrophyte (Wu et al., 2016). In wetland systems nitrogen is presented − + in the forms of organic and inorganic N (NO− 3 , NO2 , NH3 and NH4 ). They can be assimilated by submerged macrophyte, and also cause acute or chronic toxicity to aquatic organisms (Hydrilla verticillata) at high concentrations (N 1.5 mM) (Wang et al., 2010). Nitrogen is one of
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the key factors affecting the function of constructed wetlands that are dominated by submerged macrophyte. Biofilm (also known as periphyton) is a microbial community consisting of bacteria, fungi, and algae, as well as other protozoa and metazoan. These microorganisms are combined with the extracellular polymeric substances produced by the microbes in the biofilm (Flemming et al., 2007; Liu et al., 2016b). Microbes are important components of wetland systems and play an important role in the biological processes of nutrient removal through nitrification (oxidizing NH+ 4 -N to − − NO− 2 -N and NO2 -N to NO3 -N) and denitrification process, which con− vert NO− 2 -N and NO3 -N into N2 or N2O (Li et al., 2014; Hou et al., 2017). Therefore, biofilms are widely used to remove nutrients from freshwater (Pan et al., 2016; Sabater et al., 2002). Submerged macrophyte growing under water are natural substrates for the growth of biofilm and are commonly used in wetlands. Submerged macrophyte not only absorb nutrients but also provide nutrients and oxygen for biofilms (Bustamante et al., 2011). The response of plants and microorganisms to nutrition enrichment in the water column might include primary production, community structure, and the altered nutrients removal rates; each has an unique role in nutrient cycling with the biofilm (Wu et al., 2016). Respiration in dense stands of submerged vegetation at night may cause a shift from aerobic to anaerobic, which is beneficial to denitrification (Eriksson, 1999). The functional genes (narG, napA, nirS, nirK, cnorB and nosZ) related to denitrification have been detected in biofilms attached to the surface of submerged macrophyte (Potamogeton malaianus, Vallisneria natans, Ceratophyllum demersum and Elodea nuttallii) in wetlands (Zhang et al., 2016a). Biofilms are important for the removal of nutrients in wetlands, but at a high density, they may limit the growth of submerged macrophyte. Biofilms have a higher nutrient uptake rate than macrophyte and may have a negative influence on Vallisneria natans growth in eutrophic water (Song et al., 2015). The biofilm attached to leaf attenuate the incident ray received by plants and cause the decline in photosynthesis of plants (Asaeda et al., 2004). Biofilms can also affect the carbon dioxide absorption and oxygen distribution within the plants (Pang et al., 2016). Submerged plants can excrete allelopathic chemicals against microbes in biofilms, such as algae (Erhard and Gross, 2006). The bacterial and algal community was shown to be somewhat host-specific (Pang et al., 2016). However, minimal information is available about initial N loading on the growth of biofilms and nitrifiers and denitrifiers on submerged macrophyte. Abundant N is released from agricultural production systems into the surrounding water bodies, especially during the rainy seasons. Therefore, submerged macrophyte in wetlands are frequently subjected to varied N supply. In this study, a factorial experimental design was used to simulate the impact of three initial N loadings on algae, denitrifiers and nitrifiers attached to three types of submerged macrophyte: Potamogeton malaianus (PM), Vallisneria natans (VN) and Hydrilla verticillata (HV), while artificial plants (AP, abiotic plants) were used as control. The study was conducted in constructed wetland systems to test the following hypotheses: 1) N loading stimulates biofilm growth; and 2) N loading increase N-cycle genes (amoA, nirK, nirS, narG, napA and cnorB) abundance. The changes in aquatic environmental conditions, N removal, biofilm and algae densities, and six N transformation-related genes were monitored in the constructed wetlands grown with submerged macrophyte after loading with three levels of N.
were constructed with a plastic tank (V = 100 L, D = 60 cm) containing 70 L of de-chlorinated tap water and 7 cm depth of sediment. The sediment was collected from Wulongtan Lake (0–6 cm), Nanjing, China (32°02′59″ N, 118°45′47.94″ E). The concentration of key nutrients and physicochemical properties of the sediments were described in the previous study (Zhang et al., 2014). Healthy plants of P. malaianus, V. natans and H. verticillata (from Gaochun aquatic plant cultivation base, Nanjing, China, 31°55′32″ N, 118°41′47.26″ E) with similar length and biomass were selected, cleaned and then cultured in the plastic tank, where artificial plants were used as control. Biofilms attached to leaves of plants were scraped gently with a soft toothbrush as described in the paper of Pan et al. (2000). The density of the submerged macrophyte was 75 g m−2, while the artificial plants occupied 70% of the water space. The experiment was conducted in triplicate for 11 days in August and 36 tanks were used in this study. After acclimation for one week, N solution was added to the tanks to the designated levels. Fresh plant leaves were collected to examine biofilms from each tank on the 3th, 5th, 7th, 9th and 11th day after treatment with N, while pH, − DO, TN, NH+ 4 -N, and NO3 -N in the overlaying water were analyzed. 2.2. Environmental parameters and nitrogen determination Environmental parameters including pH and DO in the overlaying water were monitored using portable meters (DR2800, HACH, USA). − Concentrations of TN, NH+ 4 -N, and NO3 -N were determined using an AA3 AutoAnalyzer 3 HR (SEAL, Germany AutoAnalyzer 3 HR). 2.3. Microbe and algae densities Leaf samples (5 g) were placed into a sterile 100 mL polyethylene bottle containing 70 mL of 50 mM phosphate-buffered saline (PBS, pH = 7.4) solution. The biofilms were detached from the plant leaves in three steps: 3 min of ultra-sonication, 30 min of shaking (225 r/min), and 3 min of additional ultra-sonication (He et al., 2012). Three extracts from the same sample were combined and fixed with 2% formaldehyde. Then, 100 μL of suspension samples were stained with 700 μL of 4′,6-diamidino-2phenylindole (10 μg mL−1) in the dark for 30 min, and filtered through a 0.22 μm black membrane filter. The bacteria on the black membrane were counted under a fluorescence microscope (ZEISS, Germany). The algae were identified at the genus level at 50 random fields under a fluorescence microscope (ZEISS, Germany) with a plankton counting chamber (Beijing Purity instrument CO., LTD, China) after fixing and diluting. Detailed analysis of the steps and methods were described in a previous report (Pang et al., 2016). 2.4. Scanning electron microscopy (SEM) analysis Leaf samples collected from the four plants exposed to 12 mg L−1 TN on the 11th day were fixed with glutaraldehyde (2.5% in 50 mmol L−1 sodium cacodylate). Upon fixation, the samples were incubated in an osmium tetroxide solution (1% in 50 mmol L−1 sodium cacodylate buffer) in the dark. After immersing in a serial concentration of ethanol (in sequence: 20%, 40%, 60%, 70%, 80% and 90% ethanol) for 15 min at each concentration, the fixed samples were further dehydrated twice with 100% ethanol for 15 min. The dried samples were observed under SEM (S3400, Hitachi, Japan) after sputter coating with gold.
2. Materials and methods
2.5. DNA extraction and real time PCR amplification
2.1. Experimental design and setup
Plant samples were mixed with ethyl alcohol at 1:2 ratio (v:v) and passed through a sieve with a mesh size of 50 μm to remove plant debris, then centrifuged at 8000 rpm for 8 min. The pellets were used for DNA extraction with the Power Biofilm™ DNA Isolation kit (MoBio Laboratories, USA). Three DNA extracts from each set of samples were mixed thoroughly for further analysis. The abundance of the genes for ammonia monooxygenase (amoA), periplasmic nitrate reductase
The experiment was conducted with a 3 × 4 factorial design of three − N levels (TN = 2, 12 and 22 mg L−1, NH+ 4 -N:NO3 -N at the ratio of 1:5, TP = 0.02 mg L−1) and four plants: artificial plants (AP, a plant that is similar to the other plants and is made of non-toxic plastic), P. malaianus (PM), V. natans (VN) and H. verticillata (HV). The wetlands
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Before statistical analysis was performed, the data were checked for the assumption of normal distributions using SPSS 20 software (IBM, USA). The data of DO, pH, microbial density, algal density and the gene abundance were analyzed by one- and two-way ANOVAs using SPSS 20. Nitrogen loading and plant species were analyzed as the main factors in determining the factor effects separately by two-way ANOVAs. Redundancy analysis (RDA) was performed using Canoco 5.0 software (Microcomputer Power, USA). Cluster analysis was conducted at the algae genus level using PAST (a comprehensive statistics package). The regression analysis was performed with Microsoft Excel-2013.
Submerged macrophyte-biofilm systems can alter water pH in wetlands. In the overlaying water, pH values ranged from 7.5 (AP) to 7.85 (HV) at 2 mg L−1 TN (Fig. 1D), from 7.0 (PM) to 7.5 (VN) at 12 mg L−1 TN (Fig. 1E) and from 6.6 (PM) to 7.1 (VN) at 22 mg L−1 TN (Fig. 1F). Similarly, N loading decreased soil pH (Kennedy et al., 2005). The pH was significantly higher when exposed to 2 mg L−1 TN than 12 or 22 mg L−1 TN regardless of plants (F = 14.401, P = 0.000), but significantly higher (F = 80.323, P = 0.000) in the tanks with the submerged plants than the artificial plants. Similar to DO (F = 5.689, P = 0.000), pH (F = 3.213, P = 0.012) was significantly affected by N loading and plants species, suggesting that the differences in DO concentration and water pH can be ascribed to the interactions between plant species and N loading. A recent study (Dong et al., 2014) reported that biofilms increased the thickness of the broad diffusive boundary layer fluctuation amplitudes of DO and pH at the leaf surface of Potamogeton malaianus, while, the fluctuation amplitudes of O2 concentration and pH values declined after removing the biofilms. The photosynthetic organisms, including algae, may also contribute to the changes in the DO and pH of the overlaying water. Photoautotrophic microalgae and plants release O2 through photosynthesis and increase the pH of the water due to the absorption of dissolved carbon dioxide (Liu et al., 2016a; Schumacher et al., 2003).
3. Results and discussion
3.2. Nitrogen stimulated biofilms and algae growth
3.1. Effects of nitrogen on DO and pH of overlaying water
As revealed by SEM images (Fig. 2A–D), microbes including bacteria and algae were observed on the surface of the artificial plants and the three types of submerged macrophyte. Compared to artificial plants and P. malaianus, there were more complex structures on the leaves of V. natans and H. verticillata. Substrata architecture influenced the distribution, abundance and composition of epiphytic algae assemblages (Hinojosagarro et al., 2010), and the complexity of the leaf structure was positively correlated with the abundance and diversity of the microbial species (Sirota and Hovel, 2006). Some allelopathically active plant exudates might also affect the distribution of epiphyte biofilms (Zhang et al., 2016a). For example, phenolic substances from E. nuttallii and E. canadensis can reduce the abundance of some species in biofilms (Erhard and Gross, 2006). The microbial density increased with time and generally reached a relatively stable density between day 7 and day 9 after N loading. For the same treatment, the microbial density significantly increased with time
(napA), membrane-bound nitrate reductase (narG), nitrite reductase (nirS and nirK) and nitric oxide reductase (cnorB) was examined with the DNA samples from three biological replicates of each experiment set using their specific primer pairs, as provided in the Supplementary material (Table S1). The qPCR was conducted using a MyiQ2 real-time PCR Detection System (Bio-Rad) according to protocols described in a previous report (Zhang et al., 2016a).
2.6. Statistical analyses
Dissolved oxygen is important for aerobes in the aquatic column and increased by the presence of four plants in 11 days regardless of N level. The DO in the tanks ranged from 2.6 (AP) to 5.8 mg L− 1 (HV) at 2 mg L−1 TN, from 2.4 (AP) to 5.7 mg L−1 (VN) at 12 mg L−1 TN, and from 1.8 (PM) to 4.3 mg L−1 (VN) at 22 mg L−1 TN (Fig. 1A, B and C). Concentration of DO was significantly higher at 2 mg L−1 TN than 12 or 22 mg L−1 TN for all the plants (F = 21.238, P = 0.000) except for V. natans. The decrease in the DO level at higher N levels may be ascribed to the ammonia oxidizers and other aerobes, which consumed DO. The DO concentration was significantly higher (F = 46.021, P = 0.000) in wetlands dominated by V. natans and H. verticillata than artificial plant and P. malaianus as artificial plant does not produce O2, and leaves of P. malaianus floating on water surface, which may have reduced the exchange of O2 between water and the atmosphere. (A)
(B)
(C)
(D)
(E)
(F)
Fig. 1. Changes in dissolved oxygen (A, B and C) and pH (D, E and F) in tanks with three submerged plants and artificial plants exposed to 2, 12 and 22 mg L−1 TN, respectively over time.
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in 7 days (Fig. 2E, F and G). Consistent with our results (Fig. 2 and 3A), N loading stimulating the growth of biofilm was also reported in other studies (Gil et al., 2006; Hays, 2005). Microbial density was significantly lower in the biofilms attached to the artificial plants, as compared the three aquatic plant species (F = 4.236, P = 0.010), when exposed to 12 or 22 mg L−1 TN. According to two-way ANOVAs, microbial density was significantly affected by both N and plant species (F = 4.100, P = 0.002). The density of the algae on the 11th day in the tanks ranged from 1460 (VN) to 4200 cells cm−2 (AP) at 2 mg L−1 TN, from 1700 (HV) to 9800 cells cm−2 (AP) at 12 mg L−1 TN, and from 5100 (HV) to 16,300 cells cm−2 (AP) at 22 mg L−1 TN (Fig. 3A). The density of the algae attached to the leaves decreased among the four plants in the order of AP N PM N VN N HV. This trend was similar for all the N loading levels. Previous reports also showed that N loading stimulated the growth of algae (Gil et al., 2006; Hays, 2005), and an increase in nutrient loading in water column resulted in a substantial burst of epiphyte growth (Xie et al., 2013). The algal density was significantly higher on the artificial plants and P. malaianus than on V. natans and H. verticillata (F = 149,678.916, P = 0.000) irrespective of N levels. Nitrogen and plant species had significant effects on the density of algae (F = 23,364.294, P = 0.000). Five algal phyla (Bacillariophyta, Chlorophyta, Cyanophyta, Xanthophyta and Cryptophyta) were observed in all the samples (Table S2). Chlorophyta (66–71% and 40–64% at 2 and 12 mg L−1 TN, respectively) was the dominant phylum in the biofilms, followed by Bacillariophyta (10–14% and 21–40% at 2 and 12 mg L−1 TN, respectively). At 22 mg L−1 TN, Bacillariophyta was the dominant phylum (51–66%), followed by Chlorophyta (15–22%). Similarly, the species composition of epiphytic algae was also shown to be host-specific on submerged macrophyte Stratiotes aloides, Potamogeton lucens, Ceratophyllum demersum and Chara spp. in a lake (Toporowska et al., 2008). The algae taxa in this study were also dominated in the biofilms attached to the surface of Sparganium erectum in a shallow eutrophic turbid lake and an oligo-mesotrophic deep lake in Turkey (Albay and Akçaalan, 2008). Cluster analysis showed that three groups were generated at the genus level (Fig. 3B). Group I consisted of four plants at 22 mg L−1 TN, and group IV contained samples from V. natans and H. verticillata exposed to 12 and 2 mg L−1 TN, and group III contained samples from artificial plants and P. malaianus exposed to 12 and 2 mg L−1 TN. However, the composition of epiphytic algae was clustered in the same group at 22 mg L−1 TN
3.3. Changes in nitrogen removal and nitrogen cycle-related genes − The concentration of NH+ 4 -N (Fig. 4A, B and C), NO3 -N (Fig. 4D, E and F) and TN (Fig. 4G, H and I) decreased significantly for all treatments after 11 days. The removal rate of TN on the 11th day varied with plant species in the following order: HV (79.39%) N VA (68.85%) N PM (62.83%) N AP (62.57%) at 2 mg L−1 TN, VA (76.10%) N HV (72.91%) N PM (62.25%) N AP (51.92%) at 12 mg L−1 TN, and VA (43.58%) N HV (42.16%) N PM (35.29%) N AP (25.79%) at 22 mg L−1 TN, There were significant differ− ences in the removal of NH+ 4 -N (F = 29.026, P = 0.002), NO3 -N (F = 39.630, P = 0.002) and TN (F = 42.965, P = 0.002) among the four plant species. V. natans, H. verticillata and P. malaianus performed better in the removal of N than artificial plants regardless of N levels (Fig. 4), indicating that N was assimilated by the aquatic plants. The decrease of N in water column was mainly due to volatilization of NH+ 4 -N, uptake of plants and biofilm and denitrification process (Jing and Lin, 2004). The abundance of amoA ranged from 8.8 × 105 to 6.3 × 108 copies g−1 of fresh weight (FW) in the biofilm samples (Fig. 5A), and according to ANOVA analysis, the abundance of amoA was significantly affected by plant species (F = 11.200, P = 0.001). Abundance of amoA in the biofilms from P. malaianus, V. natans and H. verticillata plants at 12 mg L− 1 TN was significantly higher than that at 2 or 22 mg L− 1 TN, while for the artificial plants, it increased with increasing N loading. Ammonium can be oxidized into nitrite with the help of aerobic ammonium-oxidizing bacteria harboring amoA, which is a functional marker to assess the biodiversity of aerobic ammonium-oxidizing bacteria (Wang et al., 2015). A positive association exists between the potential nitrification rates and the abundance of soil ammonia-oxidizing bacteria (He et al., 2007). Nitrogen loading increased the abundance of amoA (Fig. 5A), suggesting that biofilms enhanced ammonium removal. The copy numbers of five denitrification genes, narG, napA, nirS, nirK and cnorB, were determined in the biofilm samples from the four plants exposed to the three N levels on the 11th day (Fig. 5B–F). The abundance of narG and napA genes in biofilm samples was 1.5 × 107–3.9 × 108 copies g−1 FW and 1.0 × 104–1.1 × 107 copies g−1 FW, respectively
Algae
Algae
(D)
(C)
(B)
(A)
regardless of plant species. These data suggest that the impact of N on algae composition is greater than plant species at a high N level.
Algae Algae
Bacterial aggregates Bacterial aggregates
10µm
3.91E+8
7.81E+7 1.56E+7 3.13E+6 6.25E+5 1.25E+5 2.50E+4
12 mg L-1
3.91E+8
7.81E+7
Microbial density, cells cm-2
2 mg L-1
Microbial density, cells cm-2
Microbial density, cells cm-2
(G)
(F)
(E) 3.91E+8
1.56E+7 3.13E+6 6.25E+5 1.25E+5 2.50E+4
Time, d
1.00E+3 3
5
7
9
11
P. malaianu
7.81E+7
H. verticillata 1.56E+7 3.13E+6 6.25E+5 1.25E+5 2.50E+4 5.00E+3
5.00E+3
5.00E+3
Artificial plant V. natans
22 mg L-1
Time, d
1.00E+3 3
5
7
9
11
Time, d
1.00E+3 3
5
7
9
11
Fig. 2. SEM images (A~D, 12 mg-1 TN on the 11th day) and microbial density (E, F and G) of biofilm on leaves of three submerged plants and artificial plants exposed to 2, 12 and 22 mg L−1 TN, respectively. A, artificial plant; B, P. malaianus; C, V. natans; D, H. verticillata.
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Similanty
(B)
(A) 2 mg L-1
3.1E+04
Algous density, cells cm-2
22 mg a
a b
6.3E+03
12 mg
L-1
a
a
0.54
0.60
0.66
0.78
0.84
0.90
0.96
PM22 a
b
Group
b
VN22
HV22
b
b
0.72
AP22
L-1
a
c
0.48
PM12
1.3E+03
Group
PM2 AP2
2.5E+02
AP12
Group
5.0E+01
1.0E+01
HV2
Group
Artificial plant V. natans P. malaianus H. verticillata
HV12 VN2 VN12
Fig. 3. Algal density (A) and cluster analysis (B, at genus level) of epiphytic algae attached to leaves of three submerged plants and artificial plants exposed to nitrogen on the 11th day. AP, artificial plant; PM, P. malaianus; VN, V. natans; HV, H. verticillata; 2, 12 and 22 indicate the initial TN concentration.
(Fig. 5B and C). Nitrogen loading increased napA abundance in the biofilms from the four plants. The abundance of nirK and nirS were at a range of 6.5 × 107–1.2 × 1010 copies g−1 FW (Fig. 5D) and 1.4 × 108– 2.9 × 109 copies g−1 FW (Fig. 5E), respectively. The abundance of nirS was significantly higher on P. malaianus than the other three plants (F = 36.261, P = 0.000) regardless of N level. The abundance of nirK was higher than that of nirS for the same treatment. Nitrogen loading stimulated the nirK abundance in the biofilm samples from all the plants as well as the abundance of nirS in the biofilm samples from V. natans and H. verticillata. The abundance of cnorB genes in the biofilm samples ranged from 1.9 × 106 to 2.7 × 108 copies g−1 FW (Fig. 5F). The removal rates of NO− 3 -N and TN in the tanks dominated by artificial plants was generally lower than that with the submerged macrophyte (Fig. 4D–I). In addition, the abundance of the five denitrification genes (narG, napA, nirS, qnorB, and nirK) in the artificial plants and P. (A)
(B)
malaianus was generally higher than that of V. natans and H. verticillata (Fig. 5B–F). Since artificial plants do not absorb nutrients but provide a surface for the growth of biofilms, the biofilms contribute to the removal of nitric N and total N. Among the five denitrification genes detected in this study, nitrate can be converted to nitrite by the genes narG and napA. These genes encode the subunits of two species of dissimilatory nitrate reductases Nar and Nap, respectively, while nitrite reductase encoded by nirK and nirS converts nitrite to NO or N2O, and the cnorB gene encoding cnor protein catalyzes nitric oxide reduction (Wang et al., 2015). The abundance of narG, napA, nirS, qnorB, and nirK has been investigated to explain N transformation mechanisms in wetlands (Zhang et al., 2016a). The N loading increased the abundance of the five denitrification genes, indicating that N supply increases the abundance of the corresponding denitrifiers, which contributes to the removal of N. (C)
(D)
(E)
(F)
(G)
(H)
(I)
Fig. 4. Fate of ammonia nitrogen (A, B, and C), nitric nitrogen (D, E, and F) and total nitrogen (G, H, and I) in tanks dominated by three submerged plants and artificial plants exposed to 2,12 and 22 mg L−1 TN, respectively.
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the abundance of denitrification genes was positively correlated with the concentration of NO− 3 -N and TN in this study (Fig. 6). One previous report showed that the NO− 3 removal rate was significantly correlated with the copy number of nitrite reductase genes (nirS and nirK) (Warneke et al., 2011). The concentrations of DO ranged from 1.8 to 6 mg L−1 in the overlaying water with 12 or 22 mg L−1 TN (Fig. 1A–C). The concentrations of DO ranging from 3 to 5 mg L−1 are beneficial to denitrifiers, and lower or higher concentrations of DO are usually unfavorable to the denitrification process (Ji et al., 2015). The pH values ranged from 6 to 8 mg L−1 in the overlaying water with higher N levels (12 or 22 mg L−1 TN) at the experimental stage (Fig. 1D–F). The optimal pH for aerobic denitrification is neutral to alkaline (7.0–8.0) (Ji et al., 2015). After N loading for 7 days, pH values ranged from 6 to 8 (Fig. 1D–F). These results demonstrate that during the N removal, submerged macrophyte-biofilm systems contribute to the nitrification and denitrification processes because they change pH and DO concentration that are favorable to nitrifiers and denitrifiers in wetlands (Dong et al., 2014; Ji et al., 2015).
Denitrification is associated with the abundance of the relative genes (narG, napA, nirS, nirK and nosZ) and the ratio of (napA + narG)/ (nirK + nirS) determines the accumulation of NO and N2O (Zhang et al., 2016b). In this study, the ratio (napA + narG) / (nirK + nirS) in biofilm from the artificial plants (5.6–17%) was higher than that from P. malaianus (3.8–5.6%), V. natans (0.7–0.8%) or H. verticillata (0.1– 0.6%), suggesting the three submerged plants may reduce the accumulation of NO and N2O, as compared with the artificial plants. However, the effect of submerged macrophyte-biofilm systems on the emission of the greenhouse gas N2O needs to be further studied. 3.4. Responses of biofilms to environmental conditions in constructed wetlands The impacts of environmental factors on biomass, algal density and N response-related gene abundance were analyzed using RDA (Fig. 6). Ammonium oxidizing bacteria carrying amoA catalyze ammonium and oxygen into nitrite, and this is the first step in nitrification (Ge et al., 2015). The amoA abundance was positively correlated with DO and microbial density, but negatively correlated with ammonium. Microbial density was positively correlated with DO (Fig. 6). These data demonstrate that DO and ammonium concentrations have an important role in determining the abundances of ammonium oxidizing bacteria carried amoA genes in their biofilms. Nitrate availability, DO, temperature, pH, and concentrations of TN, − NH+ 4 -N and NO3 -N are important factors limiting denitrification (Warneke et al., 2011). Similar to a previous report (Chen et al., 2014), 1.0E+10
b
1.0E+9
(A) amoA
a
a a
b
a
a
Copies g-1 fresh weight
1.0E+4 1.0E+3 1.0E+2 1.0E+1
1.0E+8
(C) nirK a a a a b b
b
1.0E+9 c
b a
1.0E+1 1.0E+0
(D) nirS a
a a a
b
b a
a b b
a
a
1.0E+8
Copies g-1 fresh weight
1.0E+6
1.0E+3 1.0E+2 1.0E+1
(E) napA
1.0E+9
2 mg L-1
1.0E+8
a
1.0E+5
b
1.0E+2 1.0E+1
1.0E+8 1.0E+7
a a
a
b
a
1.0E+4 1.0E+3 1.0E+2 1.0E+1 Artificial plant
1.0E+3
Artificial plant V. natan s P. malaianus H. verticillata
(F) cnorB
1.0E+9
22 mg L-1 b
a a
1.0E+4
1.0E+10
12 mg L-1
b
1.0E+5
1.0E+0
Artificial plant V. natan s P. malaianus H. verticillata
1.0E+10
1.0E+0
Artificial plant V. natan s P. malaianus H. verticillata
1.0E+10 1.0E+9
1.0E+4
Copies g-1 fresh weight
1.0E+2
c
1.0E+5
1.0E+6
b
1.0E+3
1.0E+7
a
a
1.0E+4
1.0E+6
1.0E+7
b
1.0E+5
1.0E+7
1.0E+0
a b b a a a
1.0E+6
Artificial plant V. natans P. malaianu s H. verticillata
a
a a a
1.0E+7
c
1.0E+10
Copies g-1 fresh weight
1.0E+8
1.0E+5
1.0E+0
1.0E+9
b
b
(B) narG
1.0E+10 a
V. natans P. malaianus H. verticillata
Copies g-1 fresh weight
Copies g-1 fresh weight
1.0E+6
Concentration of DO and pH in overlaying water of tanks decreased with increasing N loading and there were significant difference in DO concentration and water pH among the tanks with different plants. Nitrate loading stimulated biofilm growth and increased algae density. In general, the three aquatic plants have better performance in the N removal than artificial plants in the wetlands. The compositions of epiphytic algae in the biofilms at 22 mg L−1 TN were different from those
b
1.0E+8 1.0E+7
4. Conclusions
a
a a
a b
a a a
b a a
b
1.0E+6 1.0E+5 1.0E+4 1.0E+3 1.0E+2 1.0E+1 1.0E+0
Artificial plant V. natan s P. malaianus H. verticillata
Fig. 5. Relative abundances of amoA (A) and denitrification-related genes (narG, napA, nirK, nirS, cnorB, B–F) of three submerged plants and artificial plants exposed to 2, 12 and 22 mg L−1 TN. (Different lowcase letters indicates significant differences at P b 0.05).
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Fig. 6. Redundancy analysis of microbes in biofilms and environmental parameters.
at 12 or 2 mg L−1 TN. Gene amoA abundance was affected by both N level and plant species. Nitrogen loading increased the abundance of nitrifiers and denitrifiers, as revealed by the six nitrogen-cycle genes. The ratio of (napA + narG) / (nirK + nirS) in biofilm from the artificial plants was higher than that from the three submerged plants regardless of N level. These results demonstrate that submerged macrophyte-biofilm systems have an important role in N removal in wetlands. However, studies should be conducted to investigate the fate of nitrogen in the wetland dominanted with submersed macrophyte by tacking the transform of nitrogen stable isotope in the future. Competing financial interests The authors declare that there are no competing financial interests. Acknowledgments This work was financially supported by the grants from the National Natural Science Foundation of China (No. 51379063 and No. 51579075), the Jiangsu Natural Science Foundation for Excellent Youth (No. BK20160087), the Fundamental Research Funds for the Central Universities (No. 2016B06714) and a project funded through the Priority Academic Program Development of the Jiangsu Higher Education Institution. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2017.11.234. References Albay, M., Akçaalan, R., 2008. Effects of water quality and hydrologic drivers on periphyton colonization on Sparganium erectum in two Turkish lakes with different mixing regimes. Environ. Monit. Assess. 146 (1), 171–181. Asaeda, T., Sultana, M., Manatunge, J., Fujino, T., 2004. The effect of epiphytic algae on the growth and production of Potamogeton perfoliatus L. in two light conditions. Environ. Exp. Bot. 52 (3), 225–238. Bustamante, M.A., Mier, M.V., Estrada, J.A., Domíguez, C.D., 2011. Nitrogen and potassium variation on contaminant removal for a vertical subsurface flow lab scale constructed wetland. Bioresour. Technol. 102 (17), 7745–7754. Chen, Y., Wen, Y., Zhou, Q., Vymazal, J., 2014. Effects of plant biomass on denitrifying genes in subsurface-flow constructed wetlands. Bioresour. Technol. 157 (2), 341–345. De-Bashan, L.E., Bashan, Y., 2010. Immobilized microalgae for removing pollutants: review of practical aspects. Bioresour. Technol. 101 (6), 1611–1627. Dong, B., Han, R., Wang, G., Cao, X., 2014. O2, pH, and redox potential microprofiles around Potamogeton malaianus measured using microsensors. PLoS One 9 (7), e101825. Erhard, D., Gross, E.M., 2006. Allelopathic activity of Elodea canadensis and Elodea nuttallii against epiphytes and phytoplankton. Aquat. Bot. 85 (3), 203–211. Eriksson, P.G., 1999. An experimental study on effects of submersed macrophytes on nitrification and denitrification in ammonium-rich aquatic systems. Limnol. Oceanogr. 44 (8), 1993–1999. Flemming, H.C., Neu, T.R., Wozniak, D.J., 2007. The EPS matrix: the “house of biofilm cells”. J. Bacteriol. 189 (22), 7945–7947.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Nitrogen loading affects microbes, nitrifiers and denitrifiers attached to submerged macrophyte in constructed wetlands Liying Yan a, Songhe Zhang a,⁎, Da Lin a, Chuan Guo a, Lingling Yan a, Supeng Wang a, Zhenli He b a b
Ministry of Education Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, College of Environment, Hohai University, Nanjing 210098, China University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, Fort Pierce, FL 34945, USA
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Initial nitrogen supply stimulated biofilm growth and algae density. • Submerged plants performed better in the removal of nitrogen than artificial plants. • Nitrogen and the species of plant determined algae composition in biofilm. • Nitrogen loading stimulated the abundance of nitrifiers and denitrifiers.
a r t i c l e
i n f o
Article history: Received 24 October 2017 Received in revised form 19 November 2017 Accepted 20 November 2017 Available online xxxx
Keywords: Biofilms Denitrification Nitrification Submerged macrophyte Algae
a b s t r a c t Submerged macrophytes and biofilms are important components of wetlands. However, little is known about the changes of microbes in biofilms attached to submerged macrophytes upon nitrogen loading. This study investigated the changes of microbes, algae, nitrifiers and denitrifiers in biofilms attached to the leaves of artificial plants (AP), Potamogeton malaianus (PM), Vallisneria natans (VN) and Hydrilla verticillata (HV) under varied initial concentrations of total nitrogen (TN). Nitrogen addition increased biofilm biomass and changed dissolved oxygen concentrations and pH values in overlaying water. Epiphytic algal densities showed the same trend at the same N level:AP N PM N VN N HV. As revealed by cluster analysis at phylum level, algae compositions in biofilm from four plants showed some host-specific at 2 and 12 mg L−1 TN, but was clustered in the same group at 22 mg L−1 TN regardless of plant species. Submerged macrophytes had better performance in total N removal than AP. In general, N application significantly increased the abundance of amoA, nirK, nirS, napA and cnorB in biofilm. The abundance of the denitrification genes (nirK, nirS, napA, narG and cnorB) was positively correlated with nitrogen application, while amoA was correlated with concentration of dissolved oxygen. These results indicate that N loadings stimulated the growth of biofilms attached to submerged macrophyte and the removal of total N can be partially ascribed to the synergistic interactions of submerged macrophyte and biofilms in wetlands. These results highlight the ecological role of submerged macrophyte-biofilm system in nitrogen removal in wetlands. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Eutrophication of freshwater has become a global problem due to input of large amounts of nitrogen (N) and phosphorus (P) from ⁎ Corresponding author. E-mail address: [email protected] (S. Zhang).
https://doi.org/10.1016/j.scitotenv.2017.11.234 0048-9697/© 2017 Elsevier B.V. All rights reserved.
agriculture and urban areas (De-Bashan and Bashan, 2010). Eutrophication often causes algae bursts and subsequent decline of submerged macrophyte (Wu et al., 2016). In wetland systems nitrogen is presented − + in the forms of organic and inorganic N (NO− 3 , NO2 , NH3 and NH4 ). They can be assimilated by submerged macrophyte, and also cause acute or chronic toxicity to aquatic organisms (Hydrilla verticillata) at high concentrations (N 1.5 mM) (Wang et al., 2010). Nitrogen is one of
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the key factors affecting the function of constructed wetlands that are dominated by submerged macrophyte. Biofilm (also known as periphyton) is a microbial community consisting of bacteria, fungi, and algae, as well as other protozoa and metazoan. These microorganisms are combined with the extracellular polymeric substances produced by the microbes in the biofilm (Flemming et al., 2007; Liu et al., 2016b). Microbes are important components of wetland systems and play an important role in the biological processes of nutrient removal through nitrification (oxidizing NH+ 4 -N to − − NO− 2 -N and NO2 -N to NO3 -N) and denitrification process, which con− vert NO− 2 -N and NO3 -N into N2 or N2O (Li et al., 2014; Hou et al., 2017). Therefore, biofilms are widely used to remove nutrients from freshwater (Pan et al., 2016; Sabater et al., 2002). Submerged macrophyte growing under water are natural substrates for the growth of biofilm and are commonly used in wetlands. Submerged macrophyte not only absorb nutrients but also provide nutrients and oxygen for biofilms (Bustamante et al., 2011). The response of plants and microorganisms to nutrition enrichment in the water column might include primary production, community structure, and the altered nutrients removal rates; each has an unique role in nutrient cycling with the biofilm (Wu et al., 2016). Respiration in dense stands of submerged vegetation at night may cause a shift from aerobic to anaerobic, which is beneficial to denitrification (Eriksson, 1999). The functional genes (narG, napA, nirS, nirK, cnorB and nosZ) related to denitrification have been detected in biofilms attached to the surface of submerged macrophyte (Potamogeton malaianus, Vallisneria natans, Ceratophyllum demersum and Elodea nuttallii) in wetlands (Zhang et al., 2016a). Biofilms are important for the removal of nutrients in wetlands, but at a high density, they may limit the growth of submerged macrophyte. Biofilms have a higher nutrient uptake rate than macrophyte and may have a negative influence on Vallisneria natans growth in eutrophic water (Song et al., 2015). The biofilm attached to leaf attenuate the incident ray received by plants and cause the decline in photosynthesis of plants (Asaeda et al., 2004). Biofilms can also affect the carbon dioxide absorption and oxygen distribution within the plants (Pang et al., 2016). Submerged plants can excrete allelopathic chemicals against microbes in biofilms, such as algae (Erhard and Gross, 2006). The bacterial and algal community was shown to be somewhat host-specific (Pang et al., 2016). However, minimal information is available about initial N loading on the growth of biofilms and nitrifiers and denitrifiers on submerged macrophyte. Abundant N is released from agricultural production systems into the surrounding water bodies, especially during the rainy seasons. Therefore, submerged macrophyte in wetlands are frequently subjected to varied N supply. In this study, a factorial experimental design was used to simulate the impact of three initial N loadings on algae, denitrifiers and nitrifiers attached to three types of submerged macrophyte: Potamogeton malaianus (PM), Vallisneria natans (VN) and Hydrilla verticillata (HV), while artificial plants (AP, abiotic plants) were used as control. The study was conducted in constructed wetland systems to test the following hypotheses: 1) N loading stimulates biofilm growth; and 2) N loading increase N-cycle genes (amoA, nirK, nirS, narG, napA and cnorB) abundance. The changes in aquatic environmental conditions, N removal, biofilm and algae densities, and six N transformation-related genes were monitored in the constructed wetlands grown with submerged macrophyte after loading with three levels of N.
were constructed with a plastic tank (V = 100 L, D = 60 cm) containing 70 L of de-chlorinated tap water and 7 cm depth of sediment. The sediment was collected from Wulongtan Lake (0–6 cm), Nanjing, China (32°02′59″ N, 118°45′47.94″ E). The concentration of key nutrients and physicochemical properties of the sediments were described in the previous study (Zhang et al., 2014). Healthy plants of P. malaianus, V. natans and H. verticillata (from Gaochun aquatic plant cultivation base, Nanjing, China, 31°55′32″ N, 118°41′47.26″ E) with similar length and biomass were selected, cleaned and then cultured in the plastic tank, where artificial plants were used as control. Biofilms attached to leaves of plants were scraped gently with a soft toothbrush as described in the paper of Pan et al. (2000). The density of the submerged macrophyte was 75 g m−2, while the artificial plants occupied 70% of the water space. The experiment was conducted in triplicate for 11 days in August and 36 tanks were used in this study. After acclimation for one week, N solution was added to the tanks to the designated levels. Fresh plant leaves were collected to examine biofilms from each tank on the 3th, 5th, 7th, 9th and 11th day after treatment with N, while pH, − DO, TN, NH+ 4 -N, and NO3 -N in the overlaying water were analyzed. 2.2. Environmental parameters and nitrogen determination Environmental parameters including pH and DO in the overlaying water were monitored using portable meters (DR2800, HACH, USA). − Concentrations of TN, NH+ 4 -N, and NO3 -N were determined using an AA3 AutoAnalyzer 3 HR (SEAL, Germany AutoAnalyzer 3 HR). 2.3. Microbe and algae densities Leaf samples (5 g) were placed into a sterile 100 mL polyethylene bottle containing 70 mL of 50 mM phosphate-buffered saline (PBS, pH = 7.4) solution. The biofilms were detached from the plant leaves in three steps: 3 min of ultra-sonication, 30 min of shaking (225 r/min), and 3 min of additional ultra-sonication (He et al., 2012). Three extracts from the same sample were combined and fixed with 2% formaldehyde. Then, 100 μL of suspension samples were stained with 700 μL of 4′,6-diamidino-2phenylindole (10 μg mL−1) in the dark for 30 min, and filtered through a 0.22 μm black membrane filter. The bacteria on the black membrane were counted under a fluorescence microscope (ZEISS, Germany). The algae were identified at the genus level at 50 random fields under a fluorescence microscope (ZEISS, Germany) with a plankton counting chamber (Beijing Purity instrument CO., LTD, China) after fixing and diluting. Detailed analysis of the steps and methods were described in a previous report (Pang et al., 2016). 2.4. Scanning electron microscopy (SEM) analysis Leaf samples collected from the four plants exposed to 12 mg L−1 TN on the 11th day were fixed with glutaraldehyde (2.5% in 50 mmol L−1 sodium cacodylate). Upon fixation, the samples were incubated in an osmium tetroxide solution (1% in 50 mmol L−1 sodium cacodylate buffer) in the dark. After immersing in a serial concentration of ethanol (in sequence: 20%, 40%, 60%, 70%, 80% and 90% ethanol) for 15 min at each concentration, the fixed samples were further dehydrated twice with 100% ethanol for 15 min. The dried samples were observed under SEM (S3400, Hitachi, Japan) after sputter coating with gold.
2. Materials and methods
2.5. DNA extraction and real time PCR amplification
2.1. Experimental design and setup
Plant samples were mixed with ethyl alcohol at 1:2 ratio (v:v) and passed through a sieve with a mesh size of 50 μm to remove plant debris, then centrifuged at 8000 rpm for 8 min. The pellets were used for DNA extraction with the Power Biofilm™ DNA Isolation kit (MoBio Laboratories, USA). Three DNA extracts from each set of samples were mixed thoroughly for further analysis. The abundance of the genes for ammonia monooxygenase (amoA), periplasmic nitrate reductase
The experiment was conducted with a 3 × 4 factorial design of three − N levels (TN = 2, 12 and 22 mg L−1, NH+ 4 -N:NO3 -N at the ratio of 1:5, TP = 0.02 mg L−1) and four plants: artificial plants (AP, a plant that is similar to the other plants and is made of non-toxic plastic), P. malaianus (PM), V. natans (VN) and H. verticillata (HV). The wetlands
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Before statistical analysis was performed, the data were checked for the assumption of normal distributions using SPSS 20 software (IBM, USA). The data of DO, pH, microbial density, algal density and the gene abundance were analyzed by one- and two-way ANOVAs using SPSS 20. Nitrogen loading and plant species were analyzed as the main factors in determining the factor effects separately by two-way ANOVAs. Redundancy analysis (RDA) was performed using Canoco 5.0 software (Microcomputer Power, USA). Cluster analysis was conducted at the algae genus level using PAST (a comprehensive statistics package). The regression analysis was performed with Microsoft Excel-2013.
Submerged macrophyte-biofilm systems can alter water pH in wetlands. In the overlaying water, pH values ranged from 7.5 (AP) to 7.85 (HV) at 2 mg L−1 TN (Fig. 1D), from 7.0 (PM) to 7.5 (VN) at 12 mg L−1 TN (Fig. 1E) and from 6.6 (PM) to 7.1 (VN) at 22 mg L−1 TN (Fig. 1F). Similarly, N loading decreased soil pH (Kennedy et al., 2005). The pH was significantly higher when exposed to 2 mg L−1 TN than 12 or 22 mg L−1 TN regardless of plants (F = 14.401, P = 0.000), but significantly higher (F = 80.323, P = 0.000) in the tanks with the submerged plants than the artificial plants. Similar to DO (F = 5.689, P = 0.000), pH (F = 3.213, P = 0.012) was significantly affected by N loading and plants species, suggesting that the differences in DO concentration and water pH can be ascribed to the interactions between plant species and N loading. A recent study (Dong et al., 2014) reported that biofilms increased the thickness of the broad diffusive boundary layer fluctuation amplitudes of DO and pH at the leaf surface of Potamogeton malaianus, while, the fluctuation amplitudes of O2 concentration and pH values declined after removing the biofilms. The photosynthetic organisms, including algae, may also contribute to the changes in the DO and pH of the overlaying water. Photoautotrophic microalgae and plants release O2 through photosynthesis and increase the pH of the water due to the absorption of dissolved carbon dioxide (Liu et al., 2016a; Schumacher et al., 2003).
3. Results and discussion
3.2. Nitrogen stimulated biofilms and algae growth
3.1. Effects of nitrogen on DO and pH of overlaying water
As revealed by SEM images (Fig. 2A–D), microbes including bacteria and algae were observed on the surface of the artificial plants and the three types of submerged macrophyte. Compared to artificial plants and P. malaianus, there were more complex structures on the leaves of V. natans and H. verticillata. Substrata architecture influenced the distribution, abundance and composition of epiphytic algae assemblages (Hinojosagarro et al., 2010), and the complexity of the leaf structure was positively correlated with the abundance and diversity of the microbial species (Sirota and Hovel, 2006). Some allelopathically active plant exudates might also affect the distribution of epiphyte biofilms (Zhang et al., 2016a). For example, phenolic substances from E. nuttallii and E. canadensis can reduce the abundance of some species in biofilms (Erhard and Gross, 2006). The microbial density increased with time and generally reached a relatively stable density between day 7 and day 9 after N loading. For the same treatment, the microbial density significantly increased with time
(napA), membrane-bound nitrate reductase (narG), nitrite reductase (nirS and nirK) and nitric oxide reductase (cnorB) was examined with the DNA samples from three biological replicates of each experiment set using their specific primer pairs, as provided in the Supplementary material (Table S1). The qPCR was conducted using a MyiQ2 real-time PCR Detection System (Bio-Rad) according to protocols described in a previous report (Zhang et al., 2016a).
2.6. Statistical analyses
Dissolved oxygen is important for aerobes in the aquatic column and increased by the presence of four plants in 11 days regardless of N level. The DO in the tanks ranged from 2.6 (AP) to 5.8 mg L− 1 (HV) at 2 mg L−1 TN, from 2.4 (AP) to 5.7 mg L−1 (VN) at 12 mg L−1 TN, and from 1.8 (PM) to 4.3 mg L−1 (VN) at 22 mg L−1 TN (Fig. 1A, B and C). Concentration of DO was significantly higher at 2 mg L−1 TN than 12 or 22 mg L−1 TN for all the plants (F = 21.238, P = 0.000) except for V. natans. The decrease in the DO level at higher N levels may be ascribed to the ammonia oxidizers and other aerobes, which consumed DO. The DO concentration was significantly higher (F = 46.021, P = 0.000) in wetlands dominated by V. natans and H. verticillata than artificial plant and P. malaianus as artificial plant does not produce O2, and leaves of P. malaianus floating on water surface, which may have reduced the exchange of O2 between water and the atmosphere. (A)
(B)
(C)
(D)
(E)
(F)
Fig. 1. Changes in dissolved oxygen (A, B and C) and pH (D, E and F) in tanks with three submerged plants and artificial plants exposed to 2, 12 and 22 mg L−1 TN, respectively over time.
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in 7 days (Fig. 2E, F and G). Consistent with our results (Fig. 2 and 3A), N loading stimulating the growth of biofilm was also reported in other studies (Gil et al., 2006; Hays, 2005). Microbial density was significantly lower in the biofilms attached to the artificial plants, as compared the three aquatic plant species (F = 4.236, P = 0.010), when exposed to 12 or 22 mg L−1 TN. According to two-way ANOVAs, microbial density was significantly affected by both N and plant species (F = 4.100, P = 0.002). The density of the algae on the 11th day in the tanks ranged from 1460 (VN) to 4200 cells cm−2 (AP) at 2 mg L−1 TN, from 1700 (HV) to 9800 cells cm−2 (AP) at 12 mg L−1 TN, and from 5100 (HV) to 16,300 cells cm−2 (AP) at 22 mg L−1 TN (Fig. 3A). The density of the algae attached to the leaves decreased among the four plants in the order of AP N PM N VN N HV. This trend was similar for all the N loading levels. Previous reports also showed that N loading stimulated the growth of algae (Gil et al., 2006; Hays, 2005), and an increase in nutrient loading in water column resulted in a substantial burst of epiphyte growth (Xie et al., 2013). The algal density was significantly higher on the artificial plants and P. malaianus than on V. natans and H. verticillata (F = 149,678.916, P = 0.000) irrespective of N levels. Nitrogen and plant species had significant effects on the density of algae (F = 23,364.294, P = 0.000). Five algal phyla (Bacillariophyta, Chlorophyta, Cyanophyta, Xanthophyta and Cryptophyta) were observed in all the samples (Table S2). Chlorophyta (66–71% and 40–64% at 2 and 12 mg L−1 TN, respectively) was the dominant phylum in the biofilms, followed by Bacillariophyta (10–14% and 21–40% at 2 and 12 mg L−1 TN, respectively). At 22 mg L−1 TN, Bacillariophyta was the dominant phylum (51–66%), followed by Chlorophyta (15–22%). Similarly, the species composition of epiphytic algae was also shown to be host-specific on submerged macrophyte Stratiotes aloides, Potamogeton lucens, Ceratophyllum demersum and Chara spp. in a lake (Toporowska et al., 2008). The algae taxa in this study were also dominated in the biofilms attached to the surface of Sparganium erectum in a shallow eutrophic turbid lake and an oligo-mesotrophic deep lake in Turkey (Albay and Akçaalan, 2008). Cluster analysis showed that three groups were generated at the genus level (Fig. 3B). Group I consisted of four plants at 22 mg L−1 TN, and group IV contained samples from V. natans and H. verticillata exposed to 12 and 2 mg L−1 TN, and group III contained samples from artificial plants and P. malaianus exposed to 12 and 2 mg L−1 TN. However, the composition of epiphytic algae was clustered in the same group at 22 mg L−1 TN
3.3. Changes in nitrogen removal and nitrogen cycle-related genes − The concentration of NH+ 4 -N (Fig. 4A, B and C), NO3 -N (Fig. 4D, E and F) and TN (Fig. 4G, H and I) decreased significantly for all treatments after 11 days. The removal rate of TN on the 11th day varied with plant species in the following order: HV (79.39%) N VA (68.85%) N PM (62.83%) N AP (62.57%) at 2 mg L−1 TN, VA (76.10%) N HV (72.91%) N PM (62.25%) N AP (51.92%) at 12 mg L−1 TN, and VA (43.58%) N HV (42.16%) N PM (35.29%) N AP (25.79%) at 22 mg L−1 TN, There were significant differ− ences in the removal of NH+ 4 -N (F = 29.026, P = 0.002), NO3 -N (F = 39.630, P = 0.002) and TN (F = 42.965, P = 0.002) among the four plant species. V. natans, H. verticillata and P. malaianus performed better in the removal of N than artificial plants regardless of N levels (Fig. 4), indicating that N was assimilated by the aquatic plants. The decrease of N in water column was mainly due to volatilization of NH+ 4 -N, uptake of plants and biofilm and denitrification process (Jing and Lin, 2004). The abundance of amoA ranged from 8.8 × 105 to 6.3 × 108 copies g−1 of fresh weight (FW) in the biofilm samples (Fig. 5A), and according to ANOVA analysis, the abundance of amoA was significantly affected by plant species (F = 11.200, P = 0.001). Abundance of amoA in the biofilms from P. malaianus, V. natans and H. verticillata plants at 12 mg L− 1 TN was significantly higher than that at 2 or 22 mg L− 1 TN, while for the artificial plants, it increased with increasing N loading. Ammonium can be oxidized into nitrite with the help of aerobic ammonium-oxidizing bacteria harboring amoA, which is a functional marker to assess the biodiversity of aerobic ammonium-oxidizing bacteria (Wang et al., 2015). A positive association exists between the potential nitrification rates and the abundance of soil ammonia-oxidizing bacteria (He et al., 2007). Nitrogen loading increased the abundance of amoA (Fig. 5A), suggesting that biofilms enhanced ammonium removal. The copy numbers of five denitrification genes, narG, napA, nirS, nirK and cnorB, were determined in the biofilm samples from the four plants exposed to the three N levels on the 11th day (Fig. 5B–F). The abundance of narG and napA genes in biofilm samples was 1.5 × 107–3.9 × 108 copies g−1 FW and 1.0 × 104–1.1 × 107 copies g−1 FW, respectively
Algae
Algae
(D)
(C)
(B)
(A)
regardless of plant species. These data suggest that the impact of N on algae composition is greater than plant species at a high N level.
Algae Algae
Bacterial aggregates Bacterial aggregates
10µm
3.91E+8
7.81E+7 1.56E+7 3.13E+6 6.25E+5 1.25E+5 2.50E+4
12 mg L-1
3.91E+8
7.81E+7
Microbial density, cells cm-2
2 mg L-1
Microbial density, cells cm-2
Microbial density, cells cm-2
(G)
(F)
(E) 3.91E+8
1.56E+7 3.13E+6 6.25E+5 1.25E+5 2.50E+4
Time, d
1.00E+3 3
5
7
9
11
P. malaianu
7.81E+7
H. verticillata 1.56E+7 3.13E+6 6.25E+5 1.25E+5 2.50E+4 5.00E+3
5.00E+3
5.00E+3
Artificial plant V. natans
22 mg L-1
Time, d
1.00E+3 3
5
7
9
11
Time, d
1.00E+3 3
5
7
9
11
Fig. 2. SEM images (A~D, 12 mg-1 TN on the 11th day) and microbial density (E, F and G) of biofilm on leaves of three submerged plants and artificial plants exposed to 2, 12 and 22 mg L−1 TN, respectively. A, artificial plant; B, P. malaianus; C, V. natans; D, H. verticillata.
L. Yan et al. / Science of the Total Environment 622–623 (2018) 121–126
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Similanty
(B)
(A) 2 mg L-1
3.1E+04
Algous density, cells cm-2
22 mg a
a b
6.3E+03
12 mg
L-1
a
a
0.54
0.60
0.66
0.78
0.84
0.90
0.96
PM22 a
b
Group
b
VN22
HV22
b
b
0.72
AP22
L-1
a
c
0.48
PM12
1.3E+03
Group
PM2 AP2
2.5E+02
AP12
Group
5.0E+01
1.0E+01
HV2
Group
Artificial plant V. natans P. malaianus H. verticillata
HV12 VN2 VN12
Fig. 3. Algal density (A) and cluster analysis (B, at genus level) of epiphytic algae attached to leaves of three submerged plants and artificial plants exposed to nitrogen on the 11th day. AP, artificial plant; PM, P. malaianus; VN, V. natans; HV, H. verticillata; 2, 12 and 22 indicate the initial TN concentration.
(Fig. 5B and C). Nitrogen loading increased napA abundance in the biofilms from the four plants. The abundance of nirK and nirS were at a range of 6.5 × 107–1.2 × 1010 copies g−1 FW (Fig. 5D) and 1.4 × 108– 2.9 × 109 copies g−1 FW (Fig. 5E), respectively. The abundance of nirS was significantly higher on P. malaianus than the other three plants (F = 36.261, P = 0.000) regardless of N level. The abundance of nirK was higher than that of nirS for the same treatment. Nitrogen loading stimulated the nirK abundance in the biofilm samples from all the plants as well as the abundance of nirS in the biofilm samples from V. natans and H. verticillata. The abundance of cnorB genes in the biofilm samples ranged from 1.9 × 106 to 2.7 × 108 copies g−1 FW (Fig. 5F). The removal rates of NO− 3 -N and TN in the tanks dominated by artificial plants was generally lower than that with the submerged macrophyte (Fig. 4D–I). In addition, the abundance of the five denitrification genes (narG, napA, nirS, qnorB, and nirK) in the artificial plants and P. (A)
(B)
malaianus was generally higher than that of V. natans and H. verticillata (Fig. 5B–F). Since artificial plants do not absorb nutrients but provide a surface for the growth of biofilms, the biofilms contribute to the removal of nitric N and total N. Among the five denitrification genes detected in this study, nitrate can be converted to nitrite by the genes narG and napA. These genes encode the subunits of two species of dissimilatory nitrate reductases Nar and Nap, respectively, while nitrite reductase encoded by nirK and nirS converts nitrite to NO or N2O, and the cnorB gene encoding cnor protein catalyzes nitric oxide reduction (Wang et al., 2015). The abundance of narG, napA, nirS, qnorB, and nirK has been investigated to explain N transformation mechanisms in wetlands (Zhang et al., 2016a). The N loading increased the abundance of the five denitrification genes, indicating that N supply increases the abundance of the corresponding denitrifiers, which contributes to the removal of N. (C)
(D)
(E)
(F)
(G)
(H)
(I)
Fig. 4. Fate of ammonia nitrogen (A, B, and C), nitric nitrogen (D, E, and F) and total nitrogen (G, H, and I) in tanks dominated by three submerged plants and artificial plants exposed to 2,12 and 22 mg L−1 TN, respectively.
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the abundance of denitrification genes was positively correlated with the concentration of NO− 3 -N and TN in this study (Fig. 6). One previous report showed that the NO− 3 removal rate was significantly correlated with the copy number of nitrite reductase genes (nirS and nirK) (Warneke et al., 2011). The concentrations of DO ranged from 1.8 to 6 mg L−1 in the overlaying water with 12 or 22 mg L−1 TN (Fig. 1A–C). The concentrations of DO ranging from 3 to 5 mg L−1 are beneficial to denitrifiers, and lower or higher concentrations of DO are usually unfavorable to the denitrification process (Ji et al., 2015). The pH values ranged from 6 to 8 mg L−1 in the overlaying water with higher N levels (12 or 22 mg L−1 TN) at the experimental stage (Fig. 1D–F). The optimal pH for aerobic denitrification is neutral to alkaline (7.0–8.0) (Ji et al., 2015). After N loading for 7 days, pH values ranged from 6 to 8 (Fig. 1D–F). These results demonstrate that during the N removal, submerged macrophyte-biofilm systems contribute to the nitrification and denitrification processes because they change pH and DO concentration that are favorable to nitrifiers and denitrifiers in wetlands (Dong et al., 2014; Ji et al., 2015).
Denitrification is associated with the abundance of the relative genes (narG, napA, nirS, nirK and nosZ) and the ratio of (napA + narG)/ (nirK + nirS) determines the accumulation of NO and N2O (Zhang et al., 2016b). In this study, the ratio (napA + narG) / (nirK + nirS) in biofilm from the artificial plants (5.6–17%) was higher than that from P. malaianus (3.8–5.6%), V. natans (0.7–0.8%) or H. verticillata (0.1– 0.6%), suggesting the three submerged plants may reduce the accumulation of NO and N2O, as compared with the artificial plants. However, the effect of submerged macrophyte-biofilm systems on the emission of the greenhouse gas N2O needs to be further studied. 3.4. Responses of biofilms to environmental conditions in constructed wetlands The impacts of environmental factors on biomass, algal density and N response-related gene abundance were analyzed using RDA (Fig. 6). Ammonium oxidizing bacteria carrying amoA catalyze ammonium and oxygen into nitrite, and this is the first step in nitrification (Ge et al., 2015). The amoA abundance was positively correlated with DO and microbial density, but negatively correlated with ammonium. Microbial density was positively correlated with DO (Fig. 6). These data demonstrate that DO and ammonium concentrations have an important role in determining the abundances of ammonium oxidizing bacteria carried amoA genes in their biofilms. Nitrate availability, DO, temperature, pH, and concentrations of TN, − NH+ 4 -N and NO3 -N are important factors limiting denitrification (Warneke et al., 2011). Similar to a previous report (Chen et al., 2014), 1.0E+10
b
1.0E+9
(A) amoA
a
a a
b
a
a
Copies g-1 fresh weight
1.0E+4 1.0E+3 1.0E+2 1.0E+1
1.0E+8
(C) nirK a a a a b b
b
1.0E+9 c
b a
1.0E+1 1.0E+0
(D) nirS a
a a a
b
b a
a b b
a
a
1.0E+8
Copies g-1 fresh weight
1.0E+6
1.0E+3 1.0E+2 1.0E+1
(E) napA
1.0E+9
2 mg L-1
1.0E+8
a
1.0E+5
b
1.0E+2 1.0E+1
1.0E+8 1.0E+7
a a
a
b
a
1.0E+4 1.0E+3 1.0E+2 1.0E+1 Artificial plant
1.0E+3
Artificial plant V. natan s P. malaianus H. verticillata
(F) cnorB
1.0E+9
22 mg L-1 b
a a
1.0E+4
1.0E+10
12 mg L-1
b
1.0E+5
1.0E+0
Artificial plant V. natan s P. malaianus H. verticillata
1.0E+10
1.0E+0
Artificial plant V. natan s P. malaianus H. verticillata
1.0E+10 1.0E+9
1.0E+4
Copies g-1 fresh weight
1.0E+2
c
1.0E+5
1.0E+6
b
1.0E+3
1.0E+7
a
a
1.0E+4
1.0E+6
1.0E+7
b
1.0E+5
1.0E+7
1.0E+0
a b b a a a
1.0E+6
Artificial plant V. natans P. malaianu s H. verticillata
a
a a a
1.0E+7
c
1.0E+10
Copies g-1 fresh weight
1.0E+8
1.0E+5
1.0E+0
1.0E+9
b
b
(B) narG
1.0E+10 a
V. natans P. malaianus H. verticillata
Copies g-1 fresh weight
Copies g-1 fresh weight
1.0E+6
Concentration of DO and pH in overlaying water of tanks decreased with increasing N loading and there were significant difference in DO concentration and water pH among the tanks with different plants. Nitrate loading stimulated biofilm growth and increased algae density. In general, the three aquatic plants have better performance in the N removal than artificial plants in the wetlands. The compositions of epiphytic algae in the biofilms at 22 mg L−1 TN were different from those
b
1.0E+8 1.0E+7
4. Conclusions
a
a a
a b
a a a
b a a
b
1.0E+6 1.0E+5 1.0E+4 1.0E+3 1.0E+2 1.0E+1 1.0E+0
Artificial plant V. natan s P. malaianus H. verticillata
Fig. 5. Relative abundances of amoA (A) and denitrification-related genes (narG, napA, nirK, nirS, cnorB, B–F) of three submerged plants and artificial plants exposed to 2, 12 and 22 mg L−1 TN. (Different lowcase letters indicates significant differences at P b 0.05).
L. Yan et al. / Science of the Total Environment 622–623 (2018) 121–126
Fig. 6. Redundancy analysis of microbes in biofilms and environmental parameters.
at 12 or 2 mg L−1 TN. Gene amoA abundance was affected by both N level and plant species. Nitrogen loading increased the abundance of nitrifiers and denitrifiers, as revealed by the six nitrogen-cycle genes. The ratio of (napA + narG) / (nirK + nirS) in biofilm from the artificial plants was higher than that from the three submerged plants regardless of N level. These results demonstrate that submerged macrophyte-biofilm systems have an important role in N removal in wetlands. However, studies should be conducted to investigate the fate of nitrogen in the wetland dominanted with submersed macrophyte by tacking the transform of nitrogen stable isotope in the future. Competing financial interests The authors declare that there are no competing financial interests. Acknowledgments This work was financially supported by the grants from the National Natural Science Foundation of China (No. 51379063 and No. 51579075), the Jiangsu Natural Science Foundation for Excellent Youth (No. BK20160087), the Fundamental Research Funds for the Central Universities (No. 2016B06714) and a project funded through the Priority Academic Program Development of the Jiangsu Higher Education Institution. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2017.11.234. References Albay, M., Akçaalan, R., 2008. Effects of water quality and hydrologic drivers on periphyton colonization on Sparganium erectum in two Turkish lakes with different mixing regimes. Environ. Monit. Assess. 146 (1), 171–181. Asaeda, T., Sultana, M., Manatunge, J., Fujino, T., 2004. The effect of epiphytic algae on the growth and production of Potamogeton perfoliatus L. in two light conditions. Environ. Exp. Bot. 52 (3), 225–238. Bustamante, M.A., Mier, M.V., Estrada, J.A., Domíguez, C.D., 2011. Nitrogen and potassium variation on contaminant removal for a vertical subsurface flow lab scale constructed wetland. Bioresour. Technol. 102 (17), 7745–7754. Chen, Y., Wen, Y., Zhou, Q., Vymazal, J., 2014. Effects of plant biomass on denitrifying genes in subsurface-flow constructed wetlands. Bioresour. Technol. 157 (2), 341–345. De-Bashan, L.E., Bashan, Y., 2010. Immobilized microalgae for removing pollutants: review of practical aspects. Bioresour. Technol. 101 (6), 1611–1627. Dong, B., Han, R., Wang, G., Cao, X., 2014. O2, pH, and redox potential microprofiles around Potamogeton malaianus measured using microsensors. PLoS One 9 (7), e101825. Erhard, D., Gross, E.M., 2006. Allelopathic activity of Elodea canadensis and Elodea nuttallii against epiphytes and phytoplankton. Aquat. Bot. 85 (3), 203–211. Eriksson, P.G., 1999. An experimental study on effects of submersed macrophytes on nitrification and denitrification in ammonium-rich aquatic systems. Limnol. Oceanogr. 44 (8), 1993–1999. Flemming, H.C., Neu, T.R., Wozniak, D.J., 2007. The EPS matrix: the “house of biofilm cells”. J. Bacteriol. 189 (22), 7945–7947.
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