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Microbial diversity for the improvement of nitrogen removal in stormwater bioretention cells with three aquatic plants
Chemosphere 244 (2020) 125626
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Microbial diversity for the improvement of nitrogen removal in stormwater bioretention cells with three aquatic plants XiaoJun Zuo a, b, *, HongSheng Zhang a, b, Jianghua Yu a, b, ** a
Jiangsu Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, School of Environmental Science and Engineering, Nanjing University of Information Science & Technology, Nanjing, 210044, China b Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Jiangsu Joint Laboratory of Atmospheric Pollution Control, Nanjing, 210044, China
h i g h l i g h t s NH3eN removal in the bioretention cell with Lythrum salicaria L. was the highest. Nitrate removal in bioretention cells with Canna indica L. was the most significant. The used plants had different impact on top 11 dominant microflora at phylum level. Both Ramlibacter and Nitrosomonadaceae were more responsible for nitrogen removal.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2019 Received in revised form 29 November 2019 Accepted 9 December 2019 Available online 10 December 2019
The aquatic plants Iris pseudacorus L., Canna indica L. and Lythrum salicaria L. have been proved to be potential choices for nitrogen removal. However, little is known about microbial diversity for the improvement of nitrogen removal (nitrification and denitrification) in stormwater bioretention cells with the above plants. In this study, batch experiments were conducted to investigate nitrogen removal, substrate layer status, and bacterial community structure to understand microbial diversity and evaluate its effects on performances of nitrogen removal. Ammonia nitrogen removal in the bioretention cell with Lythrum salicaria L. was the highest (88.1%), which was consistent with oxidation reduction potential (ORP) in the bioretention cells. Whilst, removals for both total nitrogen and nitrate were the highest in the bioretention cell with Canna indica L., which was in line with urease activity in the mentioned cells. The used plants had different impact on top 11 dominant microflora at phylum level in the used bioretention cells. Ramlibacter and Nitrosomonadaceaea were both responsible for the difference of nitrogen removal in the bioretention cells with three aquatic plants, suggesting the enhancement of the above dominant microflora could strengthen nitrogen removal in the used bioretention cells. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: Tamara S. Galloway Keywords: Birotention Aquatic plants Nitrogen removal Oxidation reduction potential Microbial diversity
1. Introduction The rapid urbanization has increasingly changed the nature of urban underlying surface in recent years, which could shorten the
* Corresponding author. Jiangsu Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, School of Environmental Science and Engineering, Nanjing University of Information Science & Technology, Nanjing, 210044, China. ** Corresponding author. Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Jiangsu Joint Laboratory of Atmospheric Pollution Control, Nanjing, 210044, China. E-mail address: [email protected] (X. Zuo). https://doi.org/10.1016/j.chemosphere.2019.125626 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
process of generating and aggregating stormwater runoff, and further leads to urban flood, riverbank erosion and aquatic habitat destruction. Simultaneously, there were amounts of pollutants in urban stormwater runoff caused by human activities and natural processes, including suspended solids, organic carbon, nutrient, bacteria etc. (Davis and Mccuen, 2005; Ahmed et al., 2019; Rodak et al., 2019). Bioretention was one of the best management practices (BMPs) to control urban stormwater runoff, which has been widely used for the treatment of pollutants in stormwater runoff (Flanagan et al., 2018; Szota et al., 2018; Fan et al., 2019; Guo et al., 2019). The capture for both particles and particle-associated pollutants through adsorption and precipitation has been proved satisfactory,
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but removal of dissolved pollutants in stormwater runoff poses a challenge. Especially, nitrogen from stromwater runoff is one of the key factors in the eutrophication of surface water (Yan et al., 2016; Kim et al., 2019; Xu et al., 2019). It is highly soluble and does not readily adhere to bioretention media or soil, like nitrite and nitrate (Davis et al., 2006). This caused that nitrogen remained to be potential harmful to receiving water after the treatment of bioretention cells. Thus, the improvement of nitrogen removal would be the key for bioretention cells. The recent literatures have been made some successful attempts for the improvement of nitrogen removal through filling novel media (Jiang et al., 2019; Xiong et al., 2019; You et al., 2019). In our previous study (Zuo et al., 2019), a mixture with sand, soil and fly ash (1:1:1) was selected as the base in bioretention systems without plants, which was also a successful attempt. But, the role of plants in bioretention cells could be rarely found from the mentioned literatures. Plants are the important part of bioretention cells, which not only could be used for landscape, but also could directly or indirectly treat runoff pollutants (Dagenais et al., 2018; Morse et al., 2018). This implied that there could be a promising attempt for the improvement of nitrogen removal through screening different species of plants. Recently, three species of aquatic plants (including Iris pseudacorus L., Canna indica L. and Lythrum salicaria L.) have been found to be satisfactory for nutrient removal during the treatment of wastewater using constructed wetlands (Lv et al., 2016; Sun et al., 2017; Liu et al., 2019a, 2019b). It indicated that the mentioned aquatic plants could be the potential choices for the improvement of nitrogen removal in bioretention cells. But, there was only one report on nitrogen removal in bioretention cells with the used aquatic plants. Wu et al. (2017) found that the mean removal of total nitrogen (TN) from urban stormwater runoff was 49.8 ± 23.8% using biofilter with Iris pseudacorus L. and Z. matrella. Whilst, nitrogen removal in stormwater bioretention cells with Canna indica L. and Lythrum salicaria L. needs to be further investigated. Meanwhile, the comparison of nitrogen removals through stormwater bioretention cell with the mentioned plants is not systematically reported until now. Nitrogen removal in stormwater runoff by bioretention cells could be partly due to the absorption of plants, while the removal efficiencies were still limited (Dagenais et al., 2018). On the other hand, plants could provide both aerobic environment and organic compounds for the growth and reproduction of microorganisms (Minett et al., 2013; Muerdter et al., 2018), but microbial abundance or activity could change with the different of plant types (Morse et al., 2018; Payne et al., 2018). It has been widely agreed that microbial degradation was one of the important mechanisms for nitrogen removal in stormwater runoff by bioretention cells (Morse et al., 2018). Quantifying the abundance or activity of microorganisms in bioretention cells could be the key for the resolution of treatment mechanism and the improvement of nitrogen removal. Thus, the determination of microbial abundance (or activity) around plant roots in the bioretention cells would be important to improve nitrogen removal through the bioretention. Endreny et al. (2012) studied bacteria community response to saltenriched artificial stormwater through extracting DNA from column bioretention media. Similarly, Chen et al. (2013) and Waller et al. (2018) examined nitrification and denitrification genes in bioretention cells. However, there were no literatures on microbial diversity in bioretention cells with the used plants for the improvement of nitrogen removal. Therefore, sandy loam was selected as the main medium in bioretention cells with the used plants, including Iris pseudacorus L., Canna indica L. and Lythrum salicaria L., respectively, in this study. The performances of nitrogen removal in the bioretention cells with the used plants were investigated. The substrate layer status in the used bioretention cells was measured to further discuss the
difference of nitrogen removal, like soil urease activity and stromal layer oxidation reduction potential (ORP). The microbial diversity in the used bioretention cells was analyzed, as well as the effect of microbial diversity on nitrogen removal, for the improvement of nitrogen removal. 2. Materials and methods 2.1. Experimental device and operation Eight lab-scale bioretention columns were designed and placed in an open field at Nanjing University of Information science & technology, China, with number of 1-1, 1e2, 1e3, 1e4, 2-1, 2-2, 2e3, 2e4. Each bioretention column was made using the DN250 PVC pipe with 6 mm thickness and 100 cm height (Fig. 1). There was a 10 cm drain layer made by stones and pebbles at the bottom of columns (the outlet). There were two parallel bioretention columns filled using sandy loam. The control was columns (1-1, 2e1) without plants. Columns (1e2, 2-2) were installed with Iris pseudacorus L., and columns (1e3, 2e3) were installed with Canna indica L., while columns (1e4, 2e4) were installed with Lythrum salicaria L. The conditions of simulated rainfall runoff were also designed (Table 1), including interval time inflow and concentrations of pollutants in influent. A peristaltic pump (bt100-1l, China) was used to simulate rainfall events. Simulated rainfall types referred to Nanjing summer short-term heavy rainfall (Zuo et al., 2012), and the inflow concentration was set according to the quality of urban road runoff in Nanjing, China (Table 2) (Zuo et al., 2011). 2.2. Sampling and analysis methods Lab-scale bioretention experiments were to investigate nitrogen removal in the used columns, where the experiments were operated for 98 days with 7 days interval time. The total nitrogen (TN), ammonia nitrogen (NHþ 4 /NH3) and nitrate (NO3 ) in samples were determined by ion chromatography (ICS-1600, ICS-3000, and/or
Fig. 1. Pilot-scale bioretention columns.
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Table 1 Conditions of simulated rainfall runoff. Rainfall intensity (L$S1$hm2)
Designed flow (mL$min1)
Water inflow (L)
Rainfall duration (min)
Dry period days (d)
55.25
10.41
11.24
180
7
LC20, DIONEX). NHþ 4 /NH3 was detected according to the Salicylate method (Nitrogen, Ammonia, High Range, Test ‘N tube) with a detection range of 0e50 mg/L measured at 425 nm wavelength. According to the previous reference (Chen et al., 2013), the sampler was sterilized through the autoclave for 15 min at 121 C, the sample was randomly collected and mixed from five points (one at the middle and four at the surrounding) located in upper layers (10e15 cm) of filler in the used bioretention cells. Thereinto, the background samples A1 was collected on April 20th, 2018 (before running) from the control column (1-1). Four running samples (A2, A3, A4 and A5) were respectively collected from the columns (1-1, 1e2, 1e3, 1e4) on May 20th, 2018 (the unstable period). Finally, four another running samples (A6, A7, A8 and A9) were respectively collected from the columns (2e1, 2-2, 2e3, 2e4) on June 20th, 2018 (the stable period). All samples were promptly sent to the laboratory for cryopreservation, and then sent to the gene sequencing company (Majorbio, Shanghai, China) for sequencing in time. Sequencing followed the basic high-throughput sequencing process through DNA extraction, PCR amplification, and Miseq high-throughput sequencing. DNA extraction was carried out by using soil DNA extraction method. The high-throughput sequencing was completed by Illumina Miseq 2000) system, followed by OTU clustering analysis, species taxonomic analysis, and in-depth statistical data analysis about community structure and phylogenetic development. The average values of ORP in the used bioretention cells were respectively measured according to the previous literature (Martinez et al., 2018). Urease activity of the mixed samples was determined by indophenol blue colorimetry (Liu et al., 2018). All experiments were done in triplicate, and then the average of three replicate experiments were calculated and used for data interpretation. Data analysis graph was conducted by ORIGIN version 8. 3. Results and discussions 3.1. Performances of nitrogen removal The performances of nitrogen removal in the used bioretention cells were investigated respectively. It could be seen from Fig. 2 that there was little removal about TN and NO 3 in the control, which was consistent with the results on evaluation of three vegetation treatments in bioretention gardens in a semi-arid climate (Houdeshel et al., 2015). The mechanism of nitrogen removal in the bioretention cells was mainly attributed to plant absorption and microbial degradation (Bassin et al., 2011; Norton et al., 2017), which indicated that the microbial degradation was the main mechanism of nitrogen removal in the control (without plants). However, due to the poor biodegradability of the simulated stormwater (Zuo et al., 2011), it was difficult for microorganisms to
Table 2 TP, NHþ 4 /NH3, NO3 , SS and COD concentrations in the simulated stormwater. TP (mg$L1)
NH3eN (mg$L1)
NO 3 (mg$L1)
SS (mg$L1)
COD (mg$L1)
5.0
3.2
3.5
629.0
1176.0
proliferate, which resulted in the low NO 3 removal in the bioretention cells without plants. TN removal increased gradually with the running time of the used bioretention cells (Fig. 2), as well as that for both NHþ 4 /NH3 and NO 3 . However, at the unstable stage, TN removal showed a certain degree of fluctuation, as well as that for both NHþ 4 /NH3 and NO 3 . In particular, the fluctuation of nitrogen removal in the bioretention cells with Iris pseudacorus L. was the most obvious. For the bioretention cells with Iris pseudacorus L., the average removal of NHþ 4 /NH3 got to the stable (78.02%) after 5 weeks of the operation, while that of TN got to the stable (81.23%) after 9 weeks of the operation. But, NO 3 removal got to the stable (79.11%) after 10 weeks. The mentioned results were respectively close to the found with TN (35e80%), NHþ 4 /NH3 (60e80%) and NO3 (40e60%) by the bioretention cells with Iris pseudacorus L. and Z. matrella (Wu et al., 2017). However, for the bioretention cells with Canna indica L., the average removal efficiency of NO 3 got to the stable after 3 weeks with 93.90%, and that of NHþ 4 /NH3 got to the stable after 5 weeks with 83.65%, but that of TN got to the stable after 9 weeks with 86.67%, which was obviously higher than the one (85%) in greywater living walls reported by Fowdar et al. (2017). Similarly, for the bioretention cells with Lythrum salicaria L., NO 3 levels reached stability in the third week with 90.21% of the average removal efficiency, and NHþ 4 /NH3 levels reached stability in the fifth week with 88.14% of the removal efficiency. TN removal was basically stable after 9 weeks with 85.86% of the average removal efficiency, while the one (only 35e63%) of TN in pilot-scale vertical subsurface flow constructed wetlands found by Zhao et al. (2009). On the other hand, TN average removal efficiencies in the bioretention cells with the used plants after the stable were significantly higher than that (49e55%) of TN in bioretention cells with Rhododendron indicum Linnaeus claimed by Geronimo et al. (2015). However, the average removal efficiency of NH4þ/NH3 in the bioretention system with Lythrum salicaria L. was the closest to that (90%) claimed by Milandri et al. (2012). The average removal efficiencies for both TN and NO 3 were in the order of Canna indica L. > Lythrum salicaria L. > Iris pseudacorus L. > the control, while NHþ 4 /NH3 average removal efficiency was in the order of Lythrum salicaria L. > Canna indica L. > Iris pseudacorus L. > the control. Plant uptake could slightly play roles on nitrogen removal from constructed wetlands (Liu et al., 2019a, 2019b). Meanwhile, the change of microbial diversity under different plants (Dagenais et al., 2018) resulted in the varying microbial degradation, which could be the main reason for the different nitrogen removal in the used bioretention cells.
3.2. Comparisons of substrate layer status in the used bioretention cells To explore the difference of nitrogen removal in the used bioretention cells, the substrate layer status in the different cells were compared, including ORP and enzyme activity. Thereinto, as a comprehensive strength index, ORP can reflect states for both redox and aeration in soils. It could be seen from Fig. 3 that the average ORP levels in the used bioretention cells with plants were in the range of 200e300 mV, while that in the control was close to 180 mV. This implied that plants could provide a certain re-
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Fig. 2. Nitrogen removal in the used biretention systems during the operation process.
oxygenation capacity for substrate layer in the used bioretention cells, which was consistent with the found that the existence of plants could improve ORP values in simulated vertical flow constructed wetlands (Zhang et al., 2017). Meanwhile, the above results also showed that the upper layer in the used bioretention cells with plants was on the medium oxidizing status where both organic nitrogen and NHþ 4 /NH3 could be easily oxidized to NO3 (Oon et al., 2015). ORP values in the used bioretention cells were in the order of Canna indica L. > Lythrum salicaria L. > Iris pseudacorus L. > the control, which was in line with that of NHþ 4 /NH3 removal in this study. The root of Lythrum salicaria L. was the densest (S1), which was the most advantageous to the re-oxygenation of the bioretention cells. However, the one of Canna indica L. showed bulky and coarse, which was beneficial to the loosening of substrate layer. This should be the reason for the strong re-oxygenation in bioretention cells with these two plants. On the other hand, microbial enzyme activity reflected the strength and direction of various biochemical processes in soils. In particular, urease, mainly released by microbial cells during proliferation and decline processes, could stimulate the hydrolysis of peptide bonds of organic molecules (Qin et al., 2010), which had the great significance for nitrogen transformation. Meanwhile, urease was used as a comprehensive indicator of environmental suitability and activities for both microbial and plant (Liang et al., 2003), due to its sensitive to environmental factors. The urease activity in the bioretention cells with Iris pseudacorus L. approached 5 mg (g
2 h)1 after the stable, while that in the bioretention cells with Canna indica L. was the highest with 6.42 mg (g 2 h)1, which was significantly more than the one detected in constructed wetlands (Li et al., 2015). In addition, the activity of urease was 3 mg (g 2 h)1 in the bioretention cells with Lythrum salicaria L., and that in the control was the lowest with 1.45 mg (g 2 h)1. It implied that plants could promote the urease activity in substrate layer. Nitrogen conversion ability was the strongest in the bioretention cells with Canna indica L. In general, environments for the growth of both microorganisms and plants became more suitable when the enzyme activity was the higher, and thus there could be the better the performance of the system in terms of pollutant removal (Cui et al., 2013). However, it was obvious that the order of urease activity was different from that of nitrogen removals in the used bioretention cells. Although urease played an important role in microbial denitrification, the bacterial community structure (the main body of nitrification and denitrification) in the used bioretention cells with plants should be investigated clearly for the improvement of nitrogen removal. 3.3. Microbial diversity in the used bioretention cells 3.3.1. Microbial community structure In this study, a total of 1016 OTU were obtained by clustering for microbial samples from the used bioretention cells. The sample dilution graph was drawn using a sequence of number representing OTU, under the 97% similarity level (S2). It could be seen that the curve of microorganisms tended to be flat, indicating that there were reasonable sequencing amount, satisfactory sampling integrity and believable results. Chao index was an indicator for measuring the richness of microbial species (Bradley et al., 2019). It could be seen from Table 3 that the community richness in the control was obviously less than that of the used bioretention cells with plants. The richness of microbial species in the control increased firstly and then Table 3 Diversity index of the samples.
Fig. 3. The change of ORP average values at 7 days interval time in the used bioretention systems.
Samples
Shannon
Simpson
Chao
A1 A2 A3 A4 A5 A6 A7 A8 A9
5.53 4.82 5.43 5.21 4.87 4.41 4.77 5.58 5.19
0.0171 0.0241 0.0134 0.0240 0.0218 0.0406 0.0480 0.0166 0.0161
718 864 894 872 816 807 875 907 863
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decreased with the operation. Similarly, the richness of microbial species in the used bioretention cells with Iris pseudacorus L. decreased from 894 (The unstable period) to 874 (The stable period). However, the richness of microbial species increased from 872 to 907 (The bioretention cells with Canna indica L.) and from 816 to 862 (The bioretention cells with Lythrum salicaria L.). On the other hand, Shannnon index and Simpson index indicated both uniformity and diversity of microbial species in the samples, respectively (Zhao et al., 2012). The uniformity and diversity of microbial species in the control became increasingly bad with the operation. Similarly, that in the bioretention cells with Iris pseudacorus L. was getting worse. Whilst, the one in the used bioretention cells with Canna indica L. and Lythrum salicaria L. became gradually better. This indicated that Canna indica L. and Lythrum salicaria L. could promote the growth of microorganisms. However, there were different secretions and organic matters around different plant roots, which leaded to differences in the diversity and uniformity of microorganisms for different plants (Marschner et al., 2001). 3.3.2. Dominant community 3.3.2.1. Phylum level. Through the form of Sunburst pattern, the distribution of microbial species in the used bioretention cells with and without plants was analyzed. Based on Figs. 4 and S3, the top 11 dominant microflora (accounting for more than 0.1%) were found in the background sample and the running samples with the relative proportion of the total sequence exceeded 95%. But, abundances of the dominant microflora were found to be different between background sample and running samples (S3). The abundances for both Proteobacteria and Saccharibacteria in the control increased to different degrees during the operation, while that of Actinobcteria, Acidobacteria, Cyanobacteria, Nitrospirae decreased obviously. The abundance for both Firmicute and Chloroflex decreased at first and then increased at different degrees. In contrast, the one of Bacteroidetes, Verrucomicrobi and Gemmatimonadetes increased at first and then significantly decreased. On the other hand, the used plants inhibited slightly the proliferation of Proteobacteria in bioretention cells, but promoted the proliferation of Actinobacteria, Bacteroidetes, Acidobacteria, Chloroflex, Saccharibacteria, Verrucomicrobi, Gemmatimonadetes, and Cyanobacteria in different degrees. Thereinto, Iris pseudacorus L. has the most obvious promoting effect on the proliferation of Actinobacteria, while the proliferation of Acidobacteria was significantly
Fig. 4. Microbial compositions in bioretention systems at phylum level.
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promoted by Canna indica L. Lythrum salicaria L. had the most obvious promoting effect on the one for both Chloroflex and Saccharibacteria. Meanwhile, only Iris pseudacorus L. could promote the proliferation of Firmicute to a certain extent. Effects of the used plants on the proliferation of Nitrospirae were all not obvious. When the bioretention cells with three used plants reached stability, the proportion of Proteobacteria in total dominant microflora at phylum levels was the highest, but it was still lower than the one (73.3%) in pyrite constructed wetland reported by Ge et al. (2019), and different from the results on microbial structure in bioretention cells with plants including Allocasurina littoralis claimed by Morse et al. (2018). It should be attributed to plant types and water quality conditions (Payne et al., 2018).
3.3.2.2. Genus level. The bacterial community structure in the used bioretention cells at genus levels was shown in Figs. 5 and S4. There were many microbial species in the background sample, where Micrococccaceae had the largest proportion. The dominant microflora in the control, including Sphingomonas, Bacillus, Patenisporsarcina, Ramlibacter, Azotobacter, Rubelliumicrobium and Ensifer, were obviously increased during the operation. Whilst, the abundance of Massillia increased at first and then decreased during the operation, as well as that of Pedobacter, Lysobacter, Xanthomonadaceae, Oxalobacteraceae, Nocardioides, Flavisolibacter, Sphingomonadales, Altererythrobacter and Brevundimonas. Micrococcaceae, Acidobateria, Nocardioides and KD4-96 were found to decrease gradually with the operation time. In addition, Polycyclovorans disappeared after the running was started. In contrast, during the whole operation, all three used plants obstructed the proliferation of Sphingomonas, Xanthomonadacea, Oxalobacteracea, Ramlibacter, Nocardioides, Flavisolibacter, Rubellimicrobium, Sphingomonadale, Azotobacter and Ensifer in the used bioretention cells, but promoted the proliferation of Pseudomonas, Blastocatellaceae and Nitrosomonadacea in different extents. Iris pseudacorus L. was beneficial to the proliferation of Micrococcaceae. Canna indica L. was the most unfavorable to the reproduction of Bacillus, while it had a certain positive effect on the proliferation for both Massicia and fungal KD4-96. Both Iris pseudacorus L. and Canna indica L. promoted the proliferation of Acidobacteria. When the operation got to be stability, the proliferation of Paenisporosarcina was obviously promoted by Iris pseudacorus L. The abundances of Roseiflexus, Anaerolineaceae, Cytophagaceae and Saccharibacteria in the bioretention cells with Lythrum salicaria L. were all significantly found to be enhancement with the operation. All abundances of Pedobacter, Lysobacter, Altererythrobacter and Brevundimonas in unstable period were higher than that in the stable period. The proportion for both Sphingomonas and Micrococcaceae in total dominant microflora at genus levels was the highest with more than 5% in the stable bioretention cells with plants, which should be related to the found that these two genera could be widely found in contaminated soils, and they had also great survivability under low nutrient conditions (Storey et al., 2018). The proportions of Ramlibacter in total dominant microflora for the bioretention cells with Canna indica L. and Lythrum salicaria L. were close to that (4.8%) in the pyrite constructed wetland (Ge et al., 2019). The previous literature reported that the relative abundance of Gammaproteobacteria, Alphaproteobacteria, Cyanobacteria and Betaproteobacteria were dominated in all the samples from constructed wetland with Canna indica L., accounting respectively for 17.4e28.68%, 17.11e20.43%, 0.17e12.86% and 6.52e11.16% (Du et al., 2018), but there were not any such bacteria at genus levels found in stromwater bioretention cells in this study.
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Fig. 5. Microbial compositions in bioretention systems at genus level.
3.4. The relationship between microbial diversity and nitrogen removal In this study, the percentage of Proteobacteria in total dominant microflora was the highest (30.4%), and the second was the one of Actinobacteria (27.1%) and Firmicute (20.2%), after the stability of the bioretention cells with Iris pseudacorus L. However, the dominant microflora increased obviously including Proteobacteria (39.4%), Actinobacteria (20.5%), Acidobacteria (12.1%), and Chloroflex (9.5%) in the stable bioretention cells with Canna indica L. The difference about dominant microflora was found in the stable bioretention cells with Lythrum salicaria L., where there were Proteobacteria (37.5%), Actinobacteria (16.4%), Chloroflex (11.0%), Saccharibacteria (11.9%), respectively. Based on the percentage of the mentioned dominant microflora, the sum was in the order of Canna indica L. > Iris pseudacorus L. > Lythrum salicaria L., which was not in line with the order of TN, NHþ 4 /NH3 and NO3 removals. Further, the correlation analysis between nitrogen removal and proportion of dominant microflora at phylum level was made (S5). It could be found that six kinds of dominant microflora were obviously positive correlation with TN and NO 3 removals, including Actinobacteria, Acidobacteria, Chloroflex, Saccharibacteria, Verrucomicrobi and Cyanobacteria. This could explain the order of TN and NO 3 removals in the bioretention cells with plants. But, both Chloroflex and Saccharibacteria were obviously positive correlation with NHþ 4/ NH3 removals, which was in line with our previous found without plants (Zuo et al., 2019). At genus levels, after getting the stable, there were only Ramlibacter (2.1%) and Anaerolineaceae (2.5%) found in the bioretention cells with Iris pseudacorus L., while the more percentages were found in the bioretention cells with Canna indica L., including Ramlibacter (2.1%), norank_c_Acidobacteria (7.1%) and Anaerolineaceae (2.5%). However, Ramlibacter (3.3%) and Anaerolineaceae (2.6%) were found in the bioretention cells with Lythrum salicaria L. This implied that both species and abundance of dominant microflora in the used bioretention system with Canna indica L. were the most significant at genus level. Further, the correlation analysis between nitrogen removal and proportion of dominant microflora at genus level was made (S6). Results indicated that seven kinds of dominant microflora were obviously positive correlation with TN and NO 3 removals, including Saccharibacteria, Ramlibacter, Roseiflexus, KD4-
96, Nitrosomonadacea and Blastocatellaceae. However, the nitrification of nitrogen was mainly determined by Anaerolineaceae, Ramlibacter, Pedobacter and Acidobacteria (Adrados et al., 2014; Zhou et al., 2014; Zhang et al., 2018). Meanwhile, the order of Ramlibacter in abundance was in agreement with that of NHþ 4 /NH3 removal in the used bioretention cells. This suggested that NHþ 4/ NH3 removal could be attributed to Ramlibacter. Furthermore, it could be found from S6 that seven kinds of dominant microflora were obviously positive correlation with TN and NO 3 removals, including Micrococcaceae, Acidobacteria, Saccharibacteria, Roseiflexus, KD4-96, Nitrosomonadacea and Blastocatellaceae. Roseiflexus could play the main role in nitrogen denitrification, as well as Bacillus, Pseudomonas and Nitrosomonadaceae (Du et al., 2018; Wang et al., 2018; Ge et al., 2019). Thus, Roseiflexus and Nitrosomonadacea should take effect in the denitrification in this study. Both Roseiflexus (1.7%) and Nitrosomonadacea (0.9%) were found in the bioretention cells with Iris pseudacorus L. after the stable operation. Similarly, there were only two dominant microflora including Roseiflexus (2.3%) and Nitrosomonadaceae (2.1%) found in the bioretention cells with Canna indica L. But, Roseiflexus (4.2%) and Nitrosomonadaceae (0.9%) were found in the bioretention cells with Lythrum salicaria L. In terms of species and abundance, two mentioned dominant microflora involved in denitrification process was the littlest significant in bioretention cells with Iris pseudacorus L., which was in line with the removals for both TN and NO 3 in the mentioned cells. Compared with two mentioned dominant microflora in the bioretention cells with Canna indica L. and Lythrum salicaria L, although two mentioned dominant microflora was the most obvious in bioretention cells with Lythrum salicaria L., Nitrosomonadaceae in bioretention cells with Lythrum salicaria L. was significantly lower than that in bioretention cells with Canna indica L. This implied that Nitrosomonadaceae could be responsible for TN and NO 3 removals, resulting in the more satisfactory denitrification efficiency in the bioretention cells with Canna indica L. 4. Conclusions The aquatic plants Iris pseudacorus L., Canna indica L. and Lythrum salicaria L. were all found to be effective for the improvement of nutrient removal in stormwater bioretention cells in this study.
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Thereinto, NHþ 4 /NH3 removal in the bioretention cells with Lythrum salicaria L. was the highest (88.14%), which was consistent with ORP levels, while removals for both TN and nitrate NO 3 were the highest in the bioretention system with Canna indica L., which was in line with urease activity. There were the top 11 dominant microflora at phylum level, including Proteobacteria, Actinobacteria, Firmicute, Bacteroidetes, Acidobacteria, Chloroflex, Saccharibacteria, Verrucomicrobi, Gemmatimonadetes, Cyanobacteria, Nitrospirae. At genus level, Ramlibacter and Nitrosomonadaceae were found to be responsible for the difference of nitrogen removal in the bioretention cells with three aquatic plants, suggesting the enhancement of the above dominant microflora could strengthen nitrogen removal in the used bioretention cells. Author contributions section Prof. Zuo XiaoJun was in charge of the whole idea and written of this paper, meanwhile the data analysis of microbial diversity. Mr. Zhang HongSheng was in charge of Lab-scale bioretention experiments were to investigate nitrogen removal in the used columns and the data of nitrogen removal. Prof. Yu JiangHua provided part of suggestions about Section 3.4, when this paper was being revised. Declaration of competing interest None. Acknowledgments This work was supported by a grant from the project of ‘Jiangsu Specially-Appointed Professor’ (1421071801005) and Wuxi modern industrial development funding. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125626. References Adrados, B., Sanchez, O., Arias, C.A., Becares, E., Garrido, L., Mas, J., et al., 2014. Microbial communities from different types of natural wastewater treatment systems: vertical and horizontal flow constructed wetlands and biofilters. Water Res. 55, 304e312. Ahmed, W., Hamilton, K., Toze, S., Cook, S., Page, D., 2019. A review on microbial contaminants in stormwater runoff and outfalls: potential health risks and mitigation strategies. Sci. Total Environ. 692, 1304e1321. Bassin, J., Pronk, M., Kraan, R., Kleerebezem, R., Van Loosdrecht, M., 2011. Ammonium adsorption in aerobic granular sludge, activated sludge and anammox granules. Water Res. 45, 5257e5265. Bradley, I.M., Sevillano-Rivera, M.C., Pinto, A.J., Guest, J.S., 2019. Impact of solids residence time on community structure and nutrient dynamics of mixed phototrophic wastewater treatment systems. Water Res. 150, 271e282. Chen, X.L., Peltier, E., Sturm, B.S.M., Young, C.B., 2013. Nitrogen removal and nitrifying and denitrifying bacteria quantification in a stormwater bioretention system. Water Res. 47, 1691e1700. Cui, L.H., Ouyang, Y., Gu, W.J., Yang, W.Z., Xu, Q.L., 2013. Evaluation of nutrient removal efficiency and microbial enzyme activity in a baffled subsurface-flow constructed wetland system. Bioresour. Technol. 146, 656e662. Dagenais, D., Brisson, J., Fletcher, T.D., 2018. The role of plants in bioretention systems; does the science underpin current guidance? Ecol. Eng. 120, 532e545. Davis, A.P., Mccuen, R.H., 2005. Stormwater Management for Smart Growth. Springer, New York, pp. 136e141. Davis, A.P., Shokouhian, M., Sharma, H., Minami, C., 2006. Water quality improvement through bioretention media: nitrogen and phosphorus removal. Water Environ. Res. 78, 284e293. Du, L., Trinh, X.T., Chen, Q.R., Wang, C., Wang, H.H., Xia, X., et al., 2018. Enhancement of microbial nitrogen removal pathway by vegetation in Integrated VerticalFlow Constructed Wetlands (IVCWs) for treating reclaimed water. Bioresour. Technol. 249, 644e651. Endreny, T., Burke, D.J., Burchhardt, K.M., Fabian, M.W., Kretzer, A.M., 2012.
7
Bioretention column study of bacteria community response to Salt-Enriched artificial stormwater. J. Environ. Qual. 41, 1951e1959. Fan, G.D., Li, Z.S., Wang, S.M., Huang, K.S., Luo, J., 2019. Migration and transformation of nitrogen in bioretention system during rainfall runoff. Chemosphere 232, 54e62. Flanagan, K., Branchu, P., Boudahmane, L., Caupos, E., Demare, D., Deshayes, S., et al., 2018. Field performance of two biofiltration systems treating micropollutants from road runoff. Water Res. 145, 562e578. Fowdar, H.S., Hatt, B.E., Breen, P., Cook, P.L.M., Deletic, A., 2017. Designing living walls for greywater treatment. Water Res. 110, 218e232. Ge, Z.B., Wei, D.Y., Zhang, J., Hu, J.S., Liu, Z., Li, R.H., 2019. Natural pyrite to enhance simultaneous long-term nitrogen and phosphorus removal in constructed wetland: three years of pilot study. Water Res. 148, 153e161. Geronimo, F.K.F., Maniquiz-Redillas, M.C., Kim, L.H., 2015. Fate and removal of nutrients in bioretention systems. Desalin. Water Treat. 53, 3072e3079. Guo, C., Li, J.K., Li, H.E., Li, Y.J., 2019. Influences of stormwater concentration infiltration on soil nitrogen, phosphorus, TOC and their relations with enzyme activity in rain garden. Chemosphere 233, 207e215. Houdeshel, C.D., Hultine, K.R., Johnson, N.C., Pomeroy, C.A., 2015. Evaluation of three vegetation treatments in bioretention gardens in a semi-arid climate. Landsc. Urban Plan. 135, 62e72. Jiang, C.B., Li, J.K., Li, H.E., Li, Y.J., 2019. Remediation and accumulation characteristics of dissolved pollutants for stormwater in improved bioretention basins. Sci. Total Environ. 685, 763e771. Kim, M.J., Choi, S.S., Rallapalli, P.B.S., Ha, J.H., Lee, S.M., Lee, Y.S., 2019. Nitrate removal from water phase using Robinia pseudoacacia bark for solving eutrophication. Korean J. Chem. Eng. 36, 1450e1454. Liang, W., Wu, Z.B., Cheng, S.P., Zhou, Q.H., Hu, H.Y., 2003. Roles of substrate microorganisms and urease activities in wastewater purification in a constructed wetland system. Ecol. Eng. 21, 191e195. Li, J.H., Yang, X.Y., Wang, Z.F., Shan, Y., Zheng, Z., 2015. Comparison of four aquatic plant treatment systems for nutrient removal from eutrophied water. Bioresour. Technol. 179, 1e7. Liu, F.F., Fan, J.L., Du, J.H., Shi, X., Zhang, J., Shen, Y.H., 2019a. Intensified nitrogen transformation in intermittently aerated constructed wetlands: removal pathways and microbial response mechanism. Sci. Total Environ. 650, 2880e2887. Liu, T., Xu, S.R., Lu, S.Y., Qin, P., Bi, B., Ding, H.D., et al., 2019b. A review on removal of organophosphorus pesticides in constructed wetland: performance, mechanism and influencing factors. Sci. Total Environ. 651, 2247e2268. Liu, Y., Dai, Q.Y., Jin, X.Q., Dong, X.D., Peng, J., Wu, M., et al., 2018. Negative impacts of biochars on urease activity: high pH, heavy metals, polycyclic aromatic hydrocarbons, or free radicals? Environ. Sci. Technol. 52, 12740e12747. Lv, T., Zhang, Y., Zhang, L., Carvalho, P.N., Arias, C.A., Brix, H., 2016. Removal of the pesticides imazalil and tebuconazole in saturated constructed wetland mesocosms. Water Res. 91, 126e136. Marschner, P., Yang, C.H., Lieberei, R., Crowley, D.E., 2001. Soil and plant specific effects on bacterial community composition in the rhizosphere. Soil Biol. Biochem. 33, 1437e1445. Martinez, N.B., Tejeda, A., Del Toro, A., Sanchez, M.P., Zurita, F., 2018. Nitrogen removal in pilot-scale partially saturated vertical wetlands with and without an internal source of carbon. Sci. Total Environ. 645, 524e532. Milandri, S.G., Winter, K.J., Chimphango, S.B.M., Armitage, N.P., Mbui, D.N., Jackson, G.E., et al., 2012. The performance of plant species in removing nutrients from stormwater in biofiltration systems in cape town. Water S.A. 38, 655e662. Minett, D.A., Cook, P.L.M., Kessler, A.J., Cavagnaro, T.R., 2013. Root effects on the spatial and temporal dynamics of oxygen in sand-based laboratory-scale constructed biofilters. Ecol. Eng. 58, 414e422. Morse, N., Payne, E., Henry, R., Hatt, B., Chandrasena, G., Shapleigh, J., et al., 2018. Plant-microbe interactions drive denitrification rates, dissolved nitrogen removal, and the abundance of denitrification genes in stormwater control measures. Environ. Sci. Technol. 52, 9320e9329. Muerdter, C.P., Wong, C.K., LeFevre, G.H., 2018. Emerging investigator series: the role of vegetation in bioretention for stormwater treatmentin the built environment: pollutant removal, hydrologic function, and ancillary benefits. Environ. Sci. Water Res. Technol. 4, 592e612. Norton, R.A., Harrison, J.A., Keller, C.K., Moffett, K.B., 2017. Effects of storm size and frequency on nitrogen retention, denitrification, and N2O production in bioretention swale mesocosms. Biogeochemistry 134, 353e370. Oon, Y.L., Ong, S.A., Ho, L.N., Wong, Y.S., Oon, Y.S., Lehl, H.K., et al., 2015. Hybrid system up-flow constructed wetland integrated with microbial fuel cell for simultaneous wastewater treatment and electricity generation. Bioresour. Technol. 186, 270e275. Payne, E.G.I., Pham, T., Deletic, A., Hatt, B.E., Cook, P.L.M., Fletcher, T.D., 2018. Which species? A decision-support tool to guide plant selection in stormwater biofilters. Adv. Water Resour. 113, 86e99. Qin, S.P., Hu, C.S., Wang, Y.Y., Li, X.X., He, X.H., 2010. Tillage effects on intracellular and extracellular soil urease activities determined by an improved chloroform fumigation method. Soil Sci. 175, 568e572. Rodak, C.M., Moore, T.L., David, R., Jayakaran, A.D., Vogel, J.R., 2019. Urban stormwater characterization, control, and treatment. Water Environ. Res. 91, 1034e1060. Storey, S., Ashaari, M.M., Clipson, N., Doyle, E., de Menezes, A.B., 2018. Opportunistic bacteria dominate the soil microbiome response to phenanthrene in a microcosm-based study. Front. Microbiol. 9, 2815.
8
X. Zuo et al. / Chemosphere 244 (2020) 125626
Sun, W., Zhao, H.L., Wang, F., Liu, Y.F., Yang, J., Ji, M., 2017. Effect of salinity on nitrogen and phosphorus removal pathways in ahydroponic micro-ecosystem planted with Lythrum salicaria L. Ecol. Eng. 105, 205e210. Szota, C., McCarthy, M.J., Sanders, G.J., Farrell, C., Fletcher, T.D., Arndt, S.K., et al., 2018. Tree water-use strategies to improve stormwater retention performance of biofiltration systems. Water Res. 144, 285e295. Waller, L.J., Evanylo, G.K., Krometis, L.A.H., Strickland, M.S., Wynn-Thompson, T., Badgley, B.D., 2018. Engineered and environmental controls of microbial denitrification in established bioretention cells. Environ. Sci. Technol. 52, 5358e5366. Wang, R.Q., Xiao, Y.P., Lv, F.J., Hu, L.Y., Wei, L.G., Yuan, Z.Q., et al., 2018. Bacterial community structure and functional potential of rhizosphere soils as influenced by nitrogen addition and bacterial wilt disease under continuous sesame cropping. Appl. Soil Ecol. 125, 117e127. Wu, J., Cao, X.Y., Zhao, J., Dai, Y.R., Cui, N.X., Li, Z., et al., 2017. Performance of biofilter with a saturated zone for urban stormwater runoff pollution control: influence of vegetation type and saturation time. Ecol. Eng. 105, 355e361. Xiong, J.Q., Ren, S.H., He, Y.F., Wang, X.C., Bai, X.C., Wang, J.X., Dzakpasu, M., 2019. Bioretention cell incorporating Fe-biochar and saturated zones for enhanced stormwater runoff treatment. Chemosphere 237, 124424. Xu, H.L., Zhang, Y., Zhu, X.Z., Zheng, M.F., 2019. Effects of rainfall-runoff pollution on eutrophication in coastal zone: a case study in Shenzhen Bay, southern China. Nord. Hydrol 50, 1062e1075. Yan, Q., Davis, A.P., James, B.R., 2016. Enhanced organic phosphorus sorption from urban stormwater using modified bioretention media: batch studies. J. Environ. Eng. 142, 04016001. You, Z.Y., Zhang, L., Pan, S.Y., Chiang, P.C., Pei, S.L., Zhang, S.J., 2019. Performance evaluation of modified bioretention systems with alkaline solid wastes for
enhanced nutrient removal from stormwater runoff. Water Res. 161, 61e73. Zhang, C.B., Liu, W.L., Han, W.J., Guan, M., Wang, J., Liu, S.Y., et al., 2017. Responses of dissimilatory nitrate reduction to ammonium and denitrification to plant presence, plant species and species richness in simulated vertical flow constructed wetlands. Wetlands 37, 109e122. Zhang, L., Xu, K.C., Peng, Y.Z., 2018. Composition characterization and transformation mechanism of refractory dissolved organic matter from an ANAMMOX reactor fed with mature landfill leachate. Bioresour. Technol. 250, 413e421. Zhao, Y.J., Li, J.H., Wang, Z.F., Cheng, Y., Wang, S.B., Zhang, J.B., 2012. Influence of the plant development on microbial diversity of vertical-flow constructed wetlands. Biochem. Syst. Ecol. 44, 4e12. Zhao, Y.J., Liu, B., Zhang, W.G., Kong, W.J., Hu, C.W., An, S.Q., 2009. Comparison of the treatment performances of high-strength wastewater in vertical subsurface flow constructed wetlands planted with acorus calamus and Lythrum salicaria. J. Health Sci. 55, 757e766. Zhou, Z., Qiao, W.M., Xing, C., Shen, X.L., Hu, D.L., Wang, L.C., 2014. A micro-aerobic hydrolysis process for sludge in situ reduction: performance and microbial community structure. Bioresour. Technol. 173, 452e456. Zuo, X.J., Fu, D.F., Li, H., 2011. Distribution characteristics of pollutants and their mutual influence in highway runoff. Clean. - Soil, Air, Water 39, 956e963. Zuo, X.J., Fu, D.F., Li, H., 2012. Speciation distribution and mass balance of copper and zinc in urban rain, sediments, and road runoff. Environ. Sci. Pollut. Control Ser. 19, 4042e4048. Zuo, X.J., Guo, Z.Y., W, X., Yu, J.H., 2019. Diversity and metabolism effects of microorganisms in bioretention systems with sand, soil and fly ash. Sci. Total Environ. 676, 447e454.
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Microbial diversity for the improvement of nitrogen removal in stormwater bioretention cells with three aquatic plants XiaoJun Zuo a, b, *, HongSheng Zhang a, b, Jianghua Yu a, b, ** a
Jiangsu Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, School of Environmental Science and Engineering, Nanjing University of Information Science & Technology, Nanjing, 210044, China b Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Jiangsu Joint Laboratory of Atmospheric Pollution Control, Nanjing, 210044, China
h i g h l i g h t s NH3eN removal in the bioretention cell with Lythrum salicaria L. was the highest. Nitrate removal in bioretention cells with Canna indica L. was the most significant. The used plants had different impact on top 11 dominant microflora at phylum level. Both Ramlibacter and Nitrosomonadaceae were more responsible for nitrogen removal.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2019 Received in revised form 29 November 2019 Accepted 9 December 2019 Available online 10 December 2019
The aquatic plants Iris pseudacorus L., Canna indica L. and Lythrum salicaria L. have been proved to be potential choices for nitrogen removal. However, little is known about microbial diversity for the improvement of nitrogen removal (nitrification and denitrification) in stormwater bioretention cells with the above plants. In this study, batch experiments were conducted to investigate nitrogen removal, substrate layer status, and bacterial community structure to understand microbial diversity and evaluate its effects on performances of nitrogen removal. Ammonia nitrogen removal in the bioretention cell with Lythrum salicaria L. was the highest (88.1%), which was consistent with oxidation reduction potential (ORP) in the bioretention cells. Whilst, removals for both total nitrogen and nitrate were the highest in the bioretention cell with Canna indica L., which was in line with urease activity in the mentioned cells. The used plants had different impact on top 11 dominant microflora at phylum level in the used bioretention cells. Ramlibacter and Nitrosomonadaceaea were both responsible for the difference of nitrogen removal in the bioretention cells with three aquatic plants, suggesting the enhancement of the above dominant microflora could strengthen nitrogen removal in the used bioretention cells. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: Tamara S. Galloway Keywords: Birotention Aquatic plants Nitrogen removal Oxidation reduction potential Microbial diversity
1. Introduction The rapid urbanization has increasingly changed the nature of urban underlying surface in recent years, which could shorten the
* Corresponding author. Jiangsu Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, School of Environmental Science and Engineering, Nanjing University of Information Science & Technology, Nanjing, 210044, China. ** Corresponding author. Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Jiangsu Joint Laboratory of Atmospheric Pollution Control, Nanjing, 210044, China. E-mail address: [email protected] (X. Zuo). https://doi.org/10.1016/j.chemosphere.2019.125626 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
process of generating and aggregating stormwater runoff, and further leads to urban flood, riverbank erosion and aquatic habitat destruction. Simultaneously, there were amounts of pollutants in urban stormwater runoff caused by human activities and natural processes, including suspended solids, organic carbon, nutrient, bacteria etc. (Davis and Mccuen, 2005; Ahmed et al., 2019; Rodak et al., 2019). Bioretention was one of the best management practices (BMPs) to control urban stormwater runoff, which has been widely used for the treatment of pollutants in stormwater runoff (Flanagan et al., 2018; Szota et al., 2018; Fan et al., 2019; Guo et al., 2019). The capture for both particles and particle-associated pollutants through adsorption and precipitation has been proved satisfactory,
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but removal of dissolved pollutants in stormwater runoff poses a challenge. Especially, nitrogen from stromwater runoff is one of the key factors in the eutrophication of surface water (Yan et al., 2016; Kim et al., 2019; Xu et al., 2019). It is highly soluble and does not readily adhere to bioretention media or soil, like nitrite and nitrate (Davis et al., 2006). This caused that nitrogen remained to be potential harmful to receiving water after the treatment of bioretention cells. Thus, the improvement of nitrogen removal would be the key for bioretention cells. The recent literatures have been made some successful attempts for the improvement of nitrogen removal through filling novel media (Jiang et al., 2019; Xiong et al., 2019; You et al., 2019). In our previous study (Zuo et al., 2019), a mixture with sand, soil and fly ash (1:1:1) was selected as the base in bioretention systems without plants, which was also a successful attempt. But, the role of plants in bioretention cells could be rarely found from the mentioned literatures. Plants are the important part of bioretention cells, which not only could be used for landscape, but also could directly or indirectly treat runoff pollutants (Dagenais et al., 2018; Morse et al., 2018). This implied that there could be a promising attempt for the improvement of nitrogen removal through screening different species of plants. Recently, three species of aquatic plants (including Iris pseudacorus L., Canna indica L. and Lythrum salicaria L.) have been found to be satisfactory for nutrient removal during the treatment of wastewater using constructed wetlands (Lv et al., 2016; Sun et al., 2017; Liu et al., 2019a, 2019b). It indicated that the mentioned aquatic plants could be the potential choices for the improvement of nitrogen removal in bioretention cells. But, there was only one report on nitrogen removal in bioretention cells with the used aquatic plants. Wu et al. (2017) found that the mean removal of total nitrogen (TN) from urban stormwater runoff was 49.8 ± 23.8% using biofilter with Iris pseudacorus L. and Z. matrella. Whilst, nitrogen removal in stormwater bioretention cells with Canna indica L. and Lythrum salicaria L. needs to be further investigated. Meanwhile, the comparison of nitrogen removals through stormwater bioretention cell with the mentioned plants is not systematically reported until now. Nitrogen removal in stormwater runoff by bioretention cells could be partly due to the absorption of plants, while the removal efficiencies were still limited (Dagenais et al., 2018). On the other hand, plants could provide both aerobic environment and organic compounds for the growth and reproduction of microorganisms (Minett et al., 2013; Muerdter et al., 2018), but microbial abundance or activity could change with the different of plant types (Morse et al., 2018; Payne et al., 2018). It has been widely agreed that microbial degradation was one of the important mechanisms for nitrogen removal in stormwater runoff by bioretention cells (Morse et al., 2018). Quantifying the abundance or activity of microorganisms in bioretention cells could be the key for the resolution of treatment mechanism and the improvement of nitrogen removal. Thus, the determination of microbial abundance (or activity) around plant roots in the bioretention cells would be important to improve nitrogen removal through the bioretention. Endreny et al. (2012) studied bacteria community response to saltenriched artificial stormwater through extracting DNA from column bioretention media. Similarly, Chen et al. (2013) and Waller et al. (2018) examined nitrification and denitrification genes in bioretention cells. However, there were no literatures on microbial diversity in bioretention cells with the used plants for the improvement of nitrogen removal. Therefore, sandy loam was selected as the main medium in bioretention cells with the used plants, including Iris pseudacorus L., Canna indica L. and Lythrum salicaria L., respectively, in this study. The performances of nitrogen removal in the bioretention cells with the used plants were investigated. The substrate layer status in the used bioretention cells was measured to further discuss the
difference of nitrogen removal, like soil urease activity and stromal layer oxidation reduction potential (ORP). The microbial diversity in the used bioretention cells was analyzed, as well as the effect of microbial diversity on nitrogen removal, for the improvement of nitrogen removal. 2. Materials and methods 2.1. Experimental device and operation Eight lab-scale bioretention columns were designed and placed in an open field at Nanjing University of Information science & technology, China, with number of 1-1, 1e2, 1e3, 1e4, 2-1, 2-2, 2e3, 2e4. Each bioretention column was made using the DN250 PVC pipe with 6 mm thickness and 100 cm height (Fig. 1). There was a 10 cm drain layer made by stones and pebbles at the bottom of columns (the outlet). There were two parallel bioretention columns filled using sandy loam. The control was columns (1-1, 2e1) without plants. Columns (1e2, 2-2) were installed with Iris pseudacorus L., and columns (1e3, 2e3) were installed with Canna indica L., while columns (1e4, 2e4) were installed with Lythrum salicaria L. The conditions of simulated rainfall runoff were also designed (Table 1), including interval time inflow and concentrations of pollutants in influent. A peristaltic pump (bt100-1l, China) was used to simulate rainfall events. Simulated rainfall types referred to Nanjing summer short-term heavy rainfall (Zuo et al., 2012), and the inflow concentration was set according to the quality of urban road runoff in Nanjing, China (Table 2) (Zuo et al., 2011). 2.2. Sampling and analysis methods Lab-scale bioretention experiments were to investigate nitrogen removal in the used columns, where the experiments were operated for 98 days with 7 days interval time. The total nitrogen (TN), ammonia nitrogen (NHþ 4 /NH3) and nitrate (NO3 ) in samples were determined by ion chromatography (ICS-1600, ICS-3000, and/or
Fig. 1. Pilot-scale bioretention columns.
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3
Table 1 Conditions of simulated rainfall runoff. Rainfall intensity (L$S1$hm2)
Designed flow (mL$min1)
Water inflow (L)
Rainfall duration (min)
Dry period days (d)
55.25
10.41
11.24
180
7
LC20, DIONEX). NHþ 4 /NH3 was detected according to the Salicylate method (Nitrogen, Ammonia, High Range, Test ‘N tube) with a detection range of 0e50 mg/L measured at 425 nm wavelength. According to the previous reference (Chen et al., 2013), the sampler was sterilized through the autoclave for 15 min at 121 C, the sample was randomly collected and mixed from five points (one at the middle and four at the surrounding) located in upper layers (10e15 cm) of filler in the used bioretention cells. Thereinto, the background samples A1 was collected on April 20th, 2018 (before running) from the control column (1-1). Four running samples (A2, A3, A4 and A5) were respectively collected from the columns (1-1, 1e2, 1e3, 1e4) on May 20th, 2018 (the unstable period). Finally, four another running samples (A6, A7, A8 and A9) were respectively collected from the columns (2e1, 2-2, 2e3, 2e4) on June 20th, 2018 (the stable period). All samples were promptly sent to the laboratory for cryopreservation, and then sent to the gene sequencing company (Majorbio, Shanghai, China) for sequencing in time. Sequencing followed the basic high-throughput sequencing process through DNA extraction, PCR amplification, and Miseq high-throughput sequencing. DNA extraction was carried out by using soil DNA extraction method. The high-throughput sequencing was completed by Illumina Miseq 2000) system, followed by OTU clustering analysis, species taxonomic analysis, and in-depth statistical data analysis about community structure and phylogenetic development. The average values of ORP in the used bioretention cells were respectively measured according to the previous literature (Martinez et al., 2018). Urease activity of the mixed samples was determined by indophenol blue colorimetry (Liu et al., 2018). All experiments were done in triplicate, and then the average of three replicate experiments were calculated and used for data interpretation. Data analysis graph was conducted by ORIGIN version 8. 3. Results and discussions 3.1. Performances of nitrogen removal The performances of nitrogen removal in the used bioretention cells were investigated respectively. It could be seen from Fig. 2 that there was little removal about TN and NO 3 in the control, which was consistent with the results on evaluation of three vegetation treatments in bioretention gardens in a semi-arid climate (Houdeshel et al., 2015). The mechanism of nitrogen removal in the bioretention cells was mainly attributed to plant absorption and microbial degradation (Bassin et al., 2011; Norton et al., 2017), which indicated that the microbial degradation was the main mechanism of nitrogen removal in the control (without plants). However, due to the poor biodegradability of the simulated stormwater (Zuo et al., 2011), it was difficult for microorganisms to
Table 2 TP, NHþ 4 /NH3, NO3 , SS and COD concentrations in the simulated stormwater. TP (mg$L1)
NH3eN (mg$L1)
NO 3 (mg$L1)
SS (mg$L1)
COD (mg$L1)
5.0
3.2
3.5
629.0
1176.0
proliferate, which resulted in the low NO 3 removal in the bioretention cells without plants. TN removal increased gradually with the running time of the used bioretention cells (Fig. 2), as well as that for both NHþ 4 /NH3 and NO 3 . However, at the unstable stage, TN removal showed a certain degree of fluctuation, as well as that for both NHþ 4 /NH3 and NO 3 . In particular, the fluctuation of nitrogen removal in the bioretention cells with Iris pseudacorus L. was the most obvious. For the bioretention cells with Iris pseudacorus L., the average removal of NHþ 4 /NH3 got to the stable (78.02%) after 5 weeks of the operation, while that of TN got to the stable (81.23%) after 9 weeks of the operation. But, NO 3 removal got to the stable (79.11%) after 10 weeks. The mentioned results were respectively close to the found with TN (35e80%), NHþ 4 /NH3 (60e80%) and NO3 (40e60%) by the bioretention cells with Iris pseudacorus L. and Z. matrella (Wu et al., 2017). However, for the bioretention cells with Canna indica L., the average removal efficiency of NO 3 got to the stable after 3 weeks with 93.90%, and that of NHþ 4 /NH3 got to the stable after 5 weeks with 83.65%, but that of TN got to the stable after 9 weeks with 86.67%, which was obviously higher than the one (85%) in greywater living walls reported by Fowdar et al. (2017). Similarly, for the bioretention cells with Lythrum salicaria L., NO 3 levels reached stability in the third week with 90.21% of the average removal efficiency, and NHþ 4 /NH3 levels reached stability in the fifth week with 88.14% of the removal efficiency. TN removal was basically stable after 9 weeks with 85.86% of the average removal efficiency, while the one (only 35e63%) of TN in pilot-scale vertical subsurface flow constructed wetlands found by Zhao et al. (2009). On the other hand, TN average removal efficiencies in the bioretention cells with the used plants after the stable were significantly higher than that (49e55%) of TN in bioretention cells with Rhododendron indicum Linnaeus claimed by Geronimo et al. (2015). However, the average removal efficiency of NH4þ/NH3 in the bioretention system with Lythrum salicaria L. was the closest to that (90%) claimed by Milandri et al. (2012). The average removal efficiencies for both TN and NO 3 were in the order of Canna indica L. > Lythrum salicaria L. > Iris pseudacorus L. > the control, while NHþ 4 /NH3 average removal efficiency was in the order of Lythrum salicaria L. > Canna indica L. > Iris pseudacorus L. > the control. Plant uptake could slightly play roles on nitrogen removal from constructed wetlands (Liu et al., 2019a, 2019b). Meanwhile, the change of microbial diversity under different plants (Dagenais et al., 2018) resulted in the varying microbial degradation, which could be the main reason for the different nitrogen removal in the used bioretention cells.
3.2. Comparisons of substrate layer status in the used bioretention cells To explore the difference of nitrogen removal in the used bioretention cells, the substrate layer status in the different cells were compared, including ORP and enzyme activity. Thereinto, as a comprehensive strength index, ORP can reflect states for both redox and aeration in soils. It could be seen from Fig. 3 that the average ORP levels in the used bioretention cells with plants were in the range of 200e300 mV, while that in the control was close to 180 mV. This implied that plants could provide a certain re-
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Fig. 2. Nitrogen removal in the used biretention systems during the operation process.
oxygenation capacity for substrate layer in the used bioretention cells, which was consistent with the found that the existence of plants could improve ORP values in simulated vertical flow constructed wetlands (Zhang et al., 2017). Meanwhile, the above results also showed that the upper layer in the used bioretention cells with plants was on the medium oxidizing status where both organic nitrogen and NHþ 4 /NH3 could be easily oxidized to NO3 (Oon et al., 2015). ORP values in the used bioretention cells were in the order of Canna indica L. > Lythrum salicaria L. > Iris pseudacorus L. > the control, which was in line with that of NHþ 4 /NH3 removal in this study. The root of Lythrum salicaria L. was the densest (S1), which was the most advantageous to the re-oxygenation of the bioretention cells. However, the one of Canna indica L. showed bulky and coarse, which was beneficial to the loosening of substrate layer. This should be the reason for the strong re-oxygenation in bioretention cells with these two plants. On the other hand, microbial enzyme activity reflected the strength and direction of various biochemical processes in soils. In particular, urease, mainly released by microbial cells during proliferation and decline processes, could stimulate the hydrolysis of peptide bonds of organic molecules (Qin et al., 2010), which had the great significance for nitrogen transformation. Meanwhile, urease was used as a comprehensive indicator of environmental suitability and activities for both microbial and plant (Liang et al., 2003), due to its sensitive to environmental factors. The urease activity in the bioretention cells with Iris pseudacorus L. approached 5 mg (g
2 h)1 after the stable, while that in the bioretention cells with Canna indica L. was the highest with 6.42 mg (g 2 h)1, which was significantly more than the one detected in constructed wetlands (Li et al., 2015). In addition, the activity of urease was 3 mg (g 2 h)1 in the bioretention cells with Lythrum salicaria L., and that in the control was the lowest with 1.45 mg (g 2 h)1. It implied that plants could promote the urease activity in substrate layer. Nitrogen conversion ability was the strongest in the bioretention cells with Canna indica L. In general, environments for the growth of both microorganisms and plants became more suitable when the enzyme activity was the higher, and thus there could be the better the performance of the system in terms of pollutant removal (Cui et al., 2013). However, it was obvious that the order of urease activity was different from that of nitrogen removals in the used bioretention cells. Although urease played an important role in microbial denitrification, the bacterial community structure (the main body of nitrification and denitrification) in the used bioretention cells with plants should be investigated clearly for the improvement of nitrogen removal. 3.3. Microbial diversity in the used bioretention cells 3.3.1. Microbial community structure In this study, a total of 1016 OTU were obtained by clustering for microbial samples from the used bioretention cells. The sample dilution graph was drawn using a sequence of number representing OTU, under the 97% similarity level (S2). It could be seen that the curve of microorganisms tended to be flat, indicating that there were reasonable sequencing amount, satisfactory sampling integrity and believable results. Chao index was an indicator for measuring the richness of microbial species (Bradley et al., 2019). It could be seen from Table 3 that the community richness in the control was obviously less than that of the used bioretention cells with plants. The richness of microbial species in the control increased firstly and then Table 3 Diversity index of the samples.
Fig. 3. The change of ORP average values at 7 days interval time in the used bioretention systems.
Samples
Shannon
Simpson
Chao
A1 A2 A3 A4 A5 A6 A7 A8 A9
5.53 4.82 5.43 5.21 4.87 4.41 4.77 5.58 5.19
0.0171 0.0241 0.0134 0.0240 0.0218 0.0406 0.0480 0.0166 0.0161
718 864 894 872 816 807 875 907 863
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decreased with the operation. Similarly, the richness of microbial species in the used bioretention cells with Iris pseudacorus L. decreased from 894 (The unstable period) to 874 (The stable period). However, the richness of microbial species increased from 872 to 907 (The bioretention cells with Canna indica L.) and from 816 to 862 (The bioretention cells with Lythrum salicaria L.). On the other hand, Shannnon index and Simpson index indicated both uniformity and diversity of microbial species in the samples, respectively (Zhao et al., 2012). The uniformity and diversity of microbial species in the control became increasingly bad with the operation. Similarly, that in the bioretention cells with Iris pseudacorus L. was getting worse. Whilst, the one in the used bioretention cells with Canna indica L. and Lythrum salicaria L. became gradually better. This indicated that Canna indica L. and Lythrum salicaria L. could promote the growth of microorganisms. However, there were different secretions and organic matters around different plant roots, which leaded to differences in the diversity and uniformity of microorganisms for different plants (Marschner et al., 2001). 3.3.2. Dominant community 3.3.2.1. Phylum level. Through the form of Sunburst pattern, the distribution of microbial species in the used bioretention cells with and without plants was analyzed. Based on Figs. 4 and S3, the top 11 dominant microflora (accounting for more than 0.1%) were found in the background sample and the running samples with the relative proportion of the total sequence exceeded 95%. But, abundances of the dominant microflora were found to be different between background sample and running samples (S3). The abundances for both Proteobacteria and Saccharibacteria in the control increased to different degrees during the operation, while that of Actinobcteria, Acidobacteria, Cyanobacteria, Nitrospirae decreased obviously. The abundance for both Firmicute and Chloroflex decreased at first and then increased at different degrees. In contrast, the one of Bacteroidetes, Verrucomicrobi and Gemmatimonadetes increased at first and then significantly decreased. On the other hand, the used plants inhibited slightly the proliferation of Proteobacteria in bioretention cells, but promoted the proliferation of Actinobacteria, Bacteroidetes, Acidobacteria, Chloroflex, Saccharibacteria, Verrucomicrobi, Gemmatimonadetes, and Cyanobacteria in different degrees. Thereinto, Iris pseudacorus L. has the most obvious promoting effect on the proliferation of Actinobacteria, while the proliferation of Acidobacteria was significantly
Fig. 4. Microbial compositions in bioretention systems at phylum level.
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promoted by Canna indica L. Lythrum salicaria L. had the most obvious promoting effect on the one for both Chloroflex and Saccharibacteria. Meanwhile, only Iris pseudacorus L. could promote the proliferation of Firmicute to a certain extent. Effects of the used plants on the proliferation of Nitrospirae were all not obvious. When the bioretention cells with three used plants reached stability, the proportion of Proteobacteria in total dominant microflora at phylum levels was the highest, but it was still lower than the one (73.3%) in pyrite constructed wetland reported by Ge et al. (2019), and different from the results on microbial structure in bioretention cells with plants including Allocasurina littoralis claimed by Morse et al. (2018). It should be attributed to plant types and water quality conditions (Payne et al., 2018).
3.3.2.2. Genus level. The bacterial community structure in the used bioretention cells at genus levels was shown in Figs. 5 and S4. There were many microbial species in the background sample, where Micrococccaceae had the largest proportion. The dominant microflora in the control, including Sphingomonas, Bacillus, Patenisporsarcina, Ramlibacter, Azotobacter, Rubelliumicrobium and Ensifer, were obviously increased during the operation. Whilst, the abundance of Massillia increased at first and then decreased during the operation, as well as that of Pedobacter, Lysobacter, Xanthomonadaceae, Oxalobacteraceae, Nocardioides, Flavisolibacter, Sphingomonadales, Altererythrobacter and Brevundimonas. Micrococcaceae, Acidobateria, Nocardioides and KD4-96 were found to decrease gradually with the operation time. In addition, Polycyclovorans disappeared after the running was started. In contrast, during the whole operation, all three used plants obstructed the proliferation of Sphingomonas, Xanthomonadacea, Oxalobacteracea, Ramlibacter, Nocardioides, Flavisolibacter, Rubellimicrobium, Sphingomonadale, Azotobacter and Ensifer in the used bioretention cells, but promoted the proliferation of Pseudomonas, Blastocatellaceae and Nitrosomonadacea in different extents. Iris pseudacorus L. was beneficial to the proliferation of Micrococcaceae. Canna indica L. was the most unfavorable to the reproduction of Bacillus, while it had a certain positive effect on the proliferation for both Massicia and fungal KD4-96. Both Iris pseudacorus L. and Canna indica L. promoted the proliferation of Acidobacteria. When the operation got to be stability, the proliferation of Paenisporosarcina was obviously promoted by Iris pseudacorus L. The abundances of Roseiflexus, Anaerolineaceae, Cytophagaceae and Saccharibacteria in the bioretention cells with Lythrum salicaria L. were all significantly found to be enhancement with the operation. All abundances of Pedobacter, Lysobacter, Altererythrobacter and Brevundimonas in unstable period were higher than that in the stable period. The proportion for both Sphingomonas and Micrococcaceae in total dominant microflora at genus levels was the highest with more than 5% in the stable bioretention cells with plants, which should be related to the found that these two genera could be widely found in contaminated soils, and they had also great survivability under low nutrient conditions (Storey et al., 2018). The proportions of Ramlibacter in total dominant microflora for the bioretention cells with Canna indica L. and Lythrum salicaria L. were close to that (4.8%) in the pyrite constructed wetland (Ge et al., 2019). The previous literature reported that the relative abundance of Gammaproteobacteria, Alphaproteobacteria, Cyanobacteria and Betaproteobacteria were dominated in all the samples from constructed wetland with Canna indica L., accounting respectively for 17.4e28.68%, 17.11e20.43%, 0.17e12.86% and 6.52e11.16% (Du et al., 2018), but there were not any such bacteria at genus levels found in stromwater bioretention cells in this study.
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Fig. 5. Microbial compositions in bioretention systems at genus level.
3.4. The relationship between microbial diversity and nitrogen removal In this study, the percentage of Proteobacteria in total dominant microflora was the highest (30.4%), and the second was the one of Actinobacteria (27.1%) and Firmicute (20.2%), after the stability of the bioretention cells with Iris pseudacorus L. However, the dominant microflora increased obviously including Proteobacteria (39.4%), Actinobacteria (20.5%), Acidobacteria (12.1%), and Chloroflex (9.5%) in the stable bioretention cells with Canna indica L. The difference about dominant microflora was found in the stable bioretention cells with Lythrum salicaria L., where there were Proteobacteria (37.5%), Actinobacteria (16.4%), Chloroflex (11.0%), Saccharibacteria (11.9%), respectively. Based on the percentage of the mentioned dominant microflora, the sum was in the order of Canna indica L. > Iris pseudacorus L. > Lythrum salicaria L., which was not in line with the order of TN, NHþ 4 /NH3 and NO3 removals. Further, the correlation analysis between nitrogen removal and proportion of dominant microflora at phylum level was made (S5). It could be found that six kinds of dominant microflora were obviously positive correlation with TN and NO 3 removals, including Actinobacteria, Acidobacteria, Chloroflex, Saccharibacteria, Verrucomicrobi and Cyanobacteria. This could explain the order of TN and NO 3 removals in the bioretention cells with plants. But, both Chloroflex and Saccharibacteria were obviously positive correlation with NHþ 4/ NH3 removals, which was in line with our previous found without plants (Zuo et al., 2019). At genus levels, after getting the stable, there were only Ramlibacter (2.1%) and Anaerolineaceae (2.5%) found in the bioretention cells with Iris pseudacorus L., while the more percentages were found in the bioretention cells with Canna indica L., including Ramlibacter (2.1%), norank_c_Acidobacteria (7.1%) and Anaerolineaceae (2.5%). However, Ramlibacter (3.3%) and Anaerolineaceae (2.6%) were found in the bioretention cells with Lythrum salicaria L. This implied that both species and abundance of dominant microflora in the used bioretention system with Canna indica L. were the most significant at genus level. Further, the correlation analysis between nitrogen removal and proportion of dominant microflora at genus level was made (S6). Results indicated that seven kinds of dominant microflora were obviously positive correlation with TN and NO 3 removals, including Saccharibacteria, Ramlibacter, Roseiflexus, KD4-
96, Nitrosomonadacea and Blastocatellaceae. However, the nitrification of nitrogen was mainly determined by Anaerolineaceae, Ramlibacter, Pedobacter and Acidobacteria (Adrados et al., 2014; Zhou et al., 2014; Zhang et al., 2018). Meanwhile, the order of Ramlibacter in abundance was in agreement with that of NHþ 4 /NH3 removal in the used bioretention cells. This suggested that NHþ 4/ NH3 removal could be attributed to Ramlibacter. Furthermore, it could be found from S6 that seven kinds of dominant microflora were obviously positive correlation with TN and NO 3 removals, including Micrococcaceae, Acidobacteria, Saccharibacteria, Roseiflexus, KD4-96, Nitrosomonadacea and Blastocatellaceae. Roseiflexus could play the main role in nitrogen denitrification, as well as Bacillus, Pseudomonas and Nitrosomonadaceae (Du et al., 2018; Wang et al., 2018; Ge et al., 2019). Thus, Roseiflexus and Nitrosomonadacea should take effect in the denitrification in this study. Both Roseiflexus (1.7%) and Nitrosomonadacea (0.9%) were found in the bioretention cells with Iris pseudacorus L. after the stable operation. Similarly, there were only two dominant microflora including Roseiflexus (2.3%) and Nitrosomonadaceae (2.1%) found in the bioretention cells with Canna indica L. But, Roseiflexus (4.2%) and Nitrosomonadaceae (0.9%) were found in the bioretention cells with Lythrum salicaria L. In terms of species and abundance, two mentioned dominant microflora involved in denitrification process was the littlest significant in bioretention cells with Iris pseudacorus L., which was in line with the removals for both TN and NO 3 in the mentioned cells. Compared with two mentioned dominant microflora in the bioretention cells with Canna indica L. and Lythrum salicaria L, although two mentioned dominant microflora was the most obvious in bioretention cells with Lythrum salicaria L., Nitrosomonadaceae in bioretention cells with Lythrum salicaria L. was significantly lower than that in bioretention cells with Canna indica L. This implied that Nitrosomonadaceae could be responsible for TN and NO 3 removals, resulting in the more satisfactory denitrification efficiency in the bioretention cells with Canna indica L. 4. Conclusions The aquatic plants Iris pseudacorus L., Canna indica L. and Lythrum salicaria L. were all found to be effective for the improvement of nutrient removal in stormwater bioretention cells in this study.
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Thereinto, NHþ 4 /NH3 removal in the bioretention cells with Lythrum salicaria L. was the highest (88.14%), which was consistent with ORP levels, while removals for both TN and nitrate NO 3 were the highest in the bioretention system with Canna indica L., which was in line with urease activity. There were the top 11 dominant microflora at phylum level, including Proteobacteria, Actinobacteria, Firmicute, Bacteroidetes, Acidobacteria, Chloroflex, Saccharibacteria, Verrucomicrobi, Gemmatimonadetes, Cyanobacteria, Nitrospirae. At genus level, Ramlibacter and Nitrosomonadaceae were found to be responsible for the difference of nitrogen removal in the bioretention cells with three aquatic plants, suggesting the enhancement of the above dominant microflora could strengthen nitrogen removal in the used bioretention cells. Author contributions section Prof. Zuo XiaoJun was in charge of the whole idea and written of this paper, meanwhile the data analysis of microbial diversity. Mr. Zhang HongSheng was in charge of Lab-scale bioretention experiments were to investigate nitrogen removal in the used columns and the data of nitrogen removal. Prof. Yu JiangHua provided part of suggestions about Section 3.4, when this paper was being revised. Declaration of competing interest None. Acknowledgments This work was supported by a grant from the project of ‘Jiangsu Specially-Appointed Professor’ (1421071801005) and Wuxi modern industrial development funding. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125626. References Adrados, B., Sanchez, O., Arias, C.A., Becares, E., Garrido, L., Mas, J., et al., 2014. Microbial communities from different types of natural wastewater treatment systems: vertical and horizontal flow constructed wetlands and biofilters. Water Res. 55, 304e312. Ahmed, W., Hamilton, K., Toze, S., Cook, S., Page, D., 2019. A review on microbial contaminants in stormwater runoff and outfalls: potential health risks and mitigation strategies. Sci. Total Environ. 692, 1304e1321. Bassin, J., Pronk, M., Kraan, R., Kleerebezem, R., Van Loosdrecht, M., 2011. Ammonium adsorption in aerobic granular sludge, activated sludge and anammox granules. Water Res. 45, 5257e5265. Bradley, I.M., Sevillano-Rivera, M.C., Pinto, A.J., Guest, J.S., 2019. Impact of solids residence time on community structure and nutrient dynamics of mixed phototrophic wastewater treatment systems. Water Res. 150, 271e282. Chen, X.L., Peltier, E., Sturm, B.S.M., Young, C.B., 2013. Nitrogen removal and nitrifying and denitrifying bacteria quantification in a stormwater bioretention system. Water Res. 47, 1691e1700. Cui, L.H., Ouyang, Y., Gu, W.J., Yang, W.Z., Xu, Q.L., 2013. Evaluation of nutrient removal efficiency and microbial enzyme activity in a baffled subsurface-flow constructed wetland system. Bioresour. Technol. 146, 656e662. Dagenais, D., Brisson, J., Fletcher, T.D., 2018. The role of plants in bioretention systems; does the science underpin current guidance? Ecol. Eng. 120, 532e545. Davis, A.P., Mccuen, R.H., 2005. Stormwater Management for Smart Growth. Springer, New York, pp. 136e141. Davis, A.P., Shokouhian, M., Sharma, H., Minami, C., 2006. Water quality improvement through bioretention media: nitrogen and phosphorus removal. Water Environ. Res. 78, 284e293. Du, L., Trinh, X.T., Chen, Q.R., Wang, C., Wang, H.H., Xia, X., et al., 2018. Enhancement of microbial nitrogen removal pathway by vegetation in Integrated VerticalFlow Constructed Wetlands (IVCWs) for treating reclaimed water. Bioresour. Technol. 249, 644e651. Endreny, T., Burke, D.J., Burchhardt, K.M., Fabian, M.W., Kretzer, A.M., 2012.
7
Bioretention column study of bacteria community response to Salt-Enriched artificial stormwater. J. Environ. Qual. 41, 1951e1959. Fan, G.D., Li, Z.S., Wang, S.M., Huang, K.S., Luo, J., 2019. Migration and transformation of nitrogen in bioretention system during rainfall runoff. Chemosphere 232, 54e62. Flanagan, K., Branchu, P., Boudahmane, L., Caupos, E., Demare, D., Deshayes, S., et al., 2018. Field performance of two biofiltration systems treating micropollutants from road runoff. Water Res. 145, 562e578. Fowdar, H.S., Hatt, B.E., Breen, P., Cook, P.L.M., Deletic, A., 2017. Designing living walls for greywater treatment. Water Res. 110, 218e232. Ge, Z.B., Wei, D.Y., Zhang, J., Hu, J.S., Liu, Z., Li, R.H., 2019. Natural pyrite to enhance simultaneous long-term nitrogen and phosphorus removal in constructed wetland: three years of pilot study. Water Res. 148, 153e161. Geronimo, F.K.F., Maniquiz-Redillas, M.C., Kim, L.H., 2015. Fate and removal of nutrients in bioretention systems. Desalin. Water Treat. 53, 3072e3079. Guo, C., Li, J.K., Li, H.E., Li, Y.J., 2019. Influences of stormwater concentration infiltration on soil nitrogen, phosphorus, TOC and their relations with enzyme activity in rain garden. Chemosphere 233, 207e215. Houdeshel, C.D., Hultine, K.R., Johnson, N.C., Pomeroy, C.A., 2015. Evaluation of three vegetation treatments in bioretention gardens in a semi-arid climate. Landsc. Urban Plan. 135, 62e72. Jiang, C.B., Li, J.K., Li, H.E., Li, Y.J., 2019. Remediation and accumulation characteristics of dissolved pollutants for stormwater in improved bioretention basins. Sci. Total Environ. 685, 763e771. Kim, M.J., Choi, S.S., Rallapalli, P.B.S., Ha, J.H., Lee, S.M., Lee, Y.S., 2019. Nitrate removal from water phase using Robinia pseudoacacia bark for solving eutrophication. Korean J. Chem. Eng. 36, 1450e1454. Liang, W., Wu, Z.B., Cheng, S.P., Zhou, Q.H., Hu, H.Y., 2003. Roles of substrate microorganisms and urease activities in wastewater purification in a constructed wetland system. Ecol. Eng. 21, 191e195. Li, J.H., Yang, X.Y., Wang, Z.F., Shan, Y., Zheng, Z., 2015. Comparison of four aquatic plant treatment systems for nutrient removal from eutrophied water. Bioresour. Technol. 179, 1e7. Liu, F.F., Fan, J.L., Du, J.H., Shi, X., Zhang, J., Shen, Y.H., 2019a. Intensified nitrogen transformation in intermittently aerated constructed wetlands: removal pathways and microbial response mechanism. Sci. Total Environ. 650, 2880e2887. Liu, T., Xu, S.R., Lu, S.Y., Qin, P., Bi, B., Ding, H.D., et al., 2019b. A review on removal of organophosphorus pesticides in constructed wetland: performance, mechanism and influencing factors. Sci. Total Environ. 651, 2247e2268. Liu, Y., Dai, Q.Y., Jin, X.Q., Dong, X.D., Peng, J., Wu, M., et al., 2018. Negative impacts of biochars on urease activity: high pH, heavy metals, polycyclic aromatic hydrocarbons, or free radicals? Environ. Sci. Technol. 52, 12740e12747. Lv, T., Zhang, Y., Zhang, L., Carvalho, P.N., Arias, C.A., Brix, H., 2016. Removal of the pesticides imazalil and tebuconazole in saturated constructed wetland mesocosms. Water Res. 91, 126e136. Marschner, P., Yang, C.H., Lieberei, R., Crowley, D.E., 2001. Soil and plant specific effects on bacterial community composition in the rhizosphere. Soil Biol. Biochem. 33, 1437e1445. Martinez, N.B., Tejeda, A., Del Toro, A., Sanchez, M.P., Zurita, F., 2018. Nitrogen removal in pilot-scale partially saturated vertical wetlands with and without an internal source of carbon. Sci. Total Environ. 645, 524e532. Milandri, S.G., Winter, K.J., Chimphango, S.B.M., Armitage, N.P., Mbui, D.N., Jackson, G.E., et al., 2012. The performance of plant species in removing nutrients from stormwater in biofiltration systems in cape town. Water S.A. 38, 655e662. Minett, D.A., Cook, P.L.M., Kessler, A.J., Cavagnaro, T.R., 2013. Root effects on the spatial and temporal dynamics of oxygen in sand-based laboratory-scale constructed biofilters. Ecol. Eng. 58, 414e422. Morse, N., Payne, E., Henry, R., Hatt, B., Chandrasena, G., Shapleigh, J., et al., 2018. Plant-microbe interactions drive denitrification rates, dissolved nitrogen removal, and the abundance of denitrification genes in stormwater control measures. Environ. Sci. Technol. 52, 9320e9329. Muerdter, C.P., Wong, C.K., LeFevre, G.H., 2018. Emerging investigator series: the role of vegetation in bioretention for stormwater treatmentin the built environment: pollutant removal, hydrologic function, and ancillary benefits. Environ. Sci. Water Res. Technol. 4, 592e612. Norton, R.A., Harrison, J.A., Keller, C.K., Moffett, K.B., 2017. Effects of storm size and frequency on nitrogen retention, denitrification, and N2O production in bioretention swale mesocosms. Biogeochemistry 134, 353e370. Oon, Y.L., Ong, S.A., Ho, L.N., Wong, Y.S., Oon, Y.S., Lehl, H.K., et al., 2015. Hybrid system up-flow constructed wetland integrated with microbial fuel cell for simultaneous wastewater treatment and electricity generation. Bioresour. Technol. 186, 270e275. Payne, E.G.I., Pham, T., Deletic, A., Hatt, B.E., Cook, P.L.M., Fletcher, T.D., 2018. Which species? A decision-support tool to guide plant selection in stormwater biofilters. Adv. Water Resour. 113, 86e99. Qin, S.P., Hu, C.S., Wang, Y.Y., Li, X.X., He, X.H., 2010. Tillage effects on intracellular and extracellular soil urease activities determined by an improved chloroform fumigation method. Soil Sci. 175, 568e572. Rodak, C.M., Moore, T.L., David, R., Jayakaran, A.D., Vogel, J.R., 2019. Urban stormwater characterization, control, and treatment. Water Environ. Res. 91, 1034e1060. Storey, S., Ashaari, M.M., Clipson, N., Doyle, E., de Menezes, A.B., 2018. Opportunistic bacteria dominate the soil microbiome response to phenanthrene in a microcosm-based study. Front. Microbiol. 9, 2815.
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Sun, W., Zhao, H.L., Wang, F., Liu, Y.F., Yang, J., Ji, M., 2017. Effect of salinity on nitrogen and phosphorus removal pathways in ahydroponic micro-ecosystem planted with Lythrum salicaria L. Ecol. Eng. 105, 205e210. Szota, C., McCarthy, M.J., Sanders, G.J., Farrell, C., Fletcher, T.D., Arndt, S.K., et al., 2018. Tree water-use strategies to improve stormwater retention performance of biofiltration systems. Water Res. 144, 285e295. Waller, L.J., Evanylo, G.K., Krometis, L.A.H., Strickland, M.S., Wynn-Thompson, T., Badgley, B.D., 2018. Engineered and environmental controls of microbial denitrification in established bioretention cells. Environ. Sci. Technol. 52, 5358e5366. Wang, R.Q., Xiao, Y.P., Lv, F.J., Hu, L.Y., Wei, L.G., Yuan, Z.Q., et al., 2018. Bacterial community structure and functional potential of rhizosphere soils as influenced by nitrogen addition and bacterial wilt disease under continuous sesame cropping. Appl. Soil Ecol. 125, 117e127. Wu, J., Cao, X.Y., Zhao, J., Dai, Y.R., Cui, N.X., Li, Z., et al., 2017. Performance of biofilter with a saturated zone for urban stormwater runoff pollution control: influence of vegetation type and saturation time. Ecol. Eng. 105, 355e361. Xiong, J.Q., Ren, S.H., He, Y.F., Wang, X.C., Bai, X.C., Wang, J.X., Dzakpasu, M., 2019. Bioretention cell incorporating Fe-biochar and saturated zones for enhanced stormwater runoff treatment. Chemosphere 237, 124424. Xu, H.L., Zhang, Y., Zhu, X.Z., Zheng, M.F., 2019. Effects of rainfall-runoff pollution on eutrophication in coastal zone: a case study in Shenzhen Bay, southern China. Nord. Hydrol 50, 1062e1075. Yan, Q., Davis, A.P., James, B.R., 2016. Enhanced organic phosphorus sorption from urban stormwater using modified bioretention media: batch studies. J. Environ. Eng. 142, 04016001. You, Z.Y., Zhang, L., Pan, S.Y., Chiang, P.C., Pei, S.L., Zhang, S.J., 2019. Performance evaluation of modified bioretention systems with alkaline solid wastes for
enhanced nutrient removal from stormwater runoff. Water Res. 161, 61e73. Zhang, C.B., Liu, W.L., Han, W.J., Guan, M., Wang, J., Liu, S.Y., et al., 2017. Responses of dissimilatory nitrate reduction to ammonium and denitrification to plant presence, plant species and species richness in simulated vertical flow constructed wetlands. Wetlands 37, 109e122. Zhang, L., Xu, K.C., Peng, Y.Z., 2018. Composition characterization and transformation mechanism of refractory dissolved organic matter from an ANAMMOX reactor fed with mature landfill leachate. Bioresour. Technol. 250, 413e421. Zhao, Y.J., Li, J.H., Wang, Z.F., Cheng, Y., Wang, S.B., Zhang, J.B., 2012. Influence of the plant development on microbial diversity of vertical-flow constructed wetlands. Biochem. Syst. Ecol. 44, 4e12. Zhao, Y.J., Liu, B., Zhang, W.G., Kong, W.J., Hu, C.W., An, S.Q., 2009. Comparison of the treatment performances of high-strength wastewater in vertical subsurface flow constructed wetlands planted with acorus calamus and Lythrum salicaria. J. Health Sci. 55, 757e766. Zhou, Z., Qiao, W.M., Xing, C., Shen, X.L., Hu, D.L., Wang, L.C., 2014. A micro-aerobic hydrolysis process for sludge in situ reduction: performance and microbial community structure. Bioresour. Technol. 173, 452e456. Zuo, X.J., Fu, D.F., Li, H., 2011. Distribution characteristics of pollutants and their mutual influence in highway runoff. Clean. - Soil, Air, Water 39, 956e963. Zuo, X.J., Fu, D.F., Li, H., 2012. Speciation distribution and mass balance of copper and zinc in urban rain, sediments, and road runoff. Environ. Sci. Pollut. Control Ser. 19, 4042e4048. Zuo, X.J., Guo, Z.Y., W, X., Yu, J.H., 2019. Diversity and metabolism effects of microorganisms in bioretention systems with sand, soil and fly ash. Sci. Total Environ. 676, 447e454.