Evaluating wastewater stabilizing constructed wetland, through diversity and abundance of the nitrite reductase gene nirS, with regard to nitrogen control

Desalination 264 (2010) 201–205

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Desalination j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d e s a l

Evaluating wastewater stabilizing constructed wetland, through diversity and abundance of the nitrite reductase gene nirS, with regard to nitrogen control Kyongmi Chon a, Yunah Kim a, Nam Ik Chang b, Jaeweon Cho a,⁎ a

Department of Environmental Science and Engineering, NOM National Research Laboratory, Gwangju Institute of Science and Technology (GIST), Oryong-dong 1, Buk-gu,Gwangju 500-712, Republic of Korea Youngsan River Environmental Research Laboratory, 1110-8 Oryong-dong, Buk-gu, Gwangju 500-480, Republic of Korea

b

a r t i c l e

i n f o

Article history: Received 11 January 2010 Received in revised form 28 April 2010 Accepted 4 May 2010 Keywords: Constructed wetland Denitrification Nitrite reductase Real time PCR Clone library analysis

a b s t r a c t The diversity and abundance of the nitrite reductase gene nirS were investigated for a free surface flow constructed, in both the summer and winter seasons. Nitrate was efficiently removed especially by the Typha wetland, under anoxic conditions, in both seasons. Clone library analysis, which used the distance based operational taxonomic unit richness program, suggested that the Typha wetland sediment samples, collected in September, provided the highest diversity and richness, in terms of corresponding indices (Simpson D, Shannon-Weiner (H′), and Chao 1). The phylogenetic analysis based on deduced amino acids, divided 211 nirS clones into eight clusters. Although most of the clones were loosely related to the nirS gene of cultivated denitrifying bacteria, some clones were related to the nirS of Xanthomonadales, Burkholderiales, Rhodocyclales and Hydrogenophilales bacteria, over the sampling time, respectively. The real time polymerase chain reaction results showed that the copy numbers of nirS from both the Acorus and Typha wetlands in the summer (13.2 ± 2.0 and 1.8 ± 0.2 × 108copies/g soil) were significantly higher than those of the corresponding nirS genes in the winter (8.6 ± 1.1 and 0.7 ± 0.1 × 108copies/g soil). These results suggest that the diversity and abundance of the nirS can affect, in regard to nitrogen removal efficiency. © 2010 Elsevier B.V. All rights reserved.

1. Introduction A constructed wetland can be defined as an engineering technology combined with a natural system designed for wastewater control and other purposes. Constructed wetlands have been studied to efficiently control organics, nutrients, and heavy metals from either discharged water from agricultural areas or effluents from wastewater treatment plants [1–6]. When constructed wetlands have previously been used for the treatment of wastewater effluents, they have exhibited high removal efficiencies for organics (60–99%), in terms of biological oxygen demand (BOD) and chemical oxygen demand (COD), and intermediate (sometimes low) efficiencies for nutrients, in terms of ammonia, nitrate, total phosphate, etc. [2,3]. Constructed wetlands have been built to remove nitrate from wastewater effluents after conventional treatment processes, such as an activated sludge system [7]. The most important process for nitrate removal is denitrification [8], which is performed by heterotrophic bacteria. Denitrification, a microbial process in which − NO− 3 and NO2 are reduced into the gases NO, N2O and N2, plays a significant role in the nitrogen cycle. The gaseous nitrogen oxides

⁎ Corresponding author. Tel.: + 82 62 970 2443; fax. + 82 62 970 2434. E-mail address: [email protected] (J. Cho). 0011-9164/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2010.05.010

contribute not only to the green house effect but also to the depletion of stratospheric ozone layer [9]. Considering the importance of the roles of the denitrifying bacteria, those in constructed wetlands have not been well characterized. In order to investigate the diversity of denitrifying bacteria, a cyto-chrome cd1 nitrite reductase (encoded by nirS) and a copper nitrite reductase (encoded by nirK) have been used as functional gene markers, as true denitrifiers can reduce NO− 2 to gaseous nitrogen [10–12]. Specific polymerase chain reaction (PCR) primer sets for these genes have been used to study the structure of the denitrifying community and to quantify denitrifying bacteria from various environmental samples, using cloning library methods and real time PCR, respectively [13–19]. Our previous studies [19] revealed that nirS functional genes were the most dominant in the effluent-fed wetland samples. With that, nirS was selected as a functional gene marker to identify diversity and abundance of denitrifiers in constructed wetland soils. An investigation of the diversity of denitrifying bacteria and changes in the denitrifying community structure that occur due to natural and anthropogenic effects on wastewater treatment processes, is believed necessary to better comprehend the roles of denitrifying bacteria in nitrogen cycling. The objectives of this study were to investigate the denitrifying community structure in constructed wetland sediments through the analysis of nirS functional genes, and to monitor seasonal changes in the diversity and quantity of nirS genes with respect to controls of nitrate in constructed wetlands.

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2. Materials and methods 2.1. Site description and sampling Wastewater effluent, treated by secondary wastewater processes, and wetland samples were collected from the Damyang wastewater treatment plant and adjacent free surface-flow constructed wetlands (35°18′N, 126°58′E), respectively. The wetlands involved two different ponds, with Acorus and Typha plants (i.e., wastewater treatment plant → Acorus pond → Typha pond → wetland effluent). The flow rate and hydraulic retention time of the whole wetlands were designed to be approximately 1800 m3/day and 6 h, respectively. The average length, width, and depth of the whole wetlands were ca. 120, 30, and 0.13 m, respectively [6]. Field water and soil samplings were conducted on September 3rd and November 17th, 2009. The oxidation reduction potential (ORP), pH, and temperature of each − sample were measured in the field. Both N–NO− 2 and N–NO3 concentrations of the samples were measured using an ion chromatography (IC) apparatus (DX-120, Dionex, CA, US), equipped with an AS14 column (4× 250 mm, Dionex, CA, US). The dissolved organic carbon (DOC) and total nitrogen (TN) of the wastewater and wetlands samples were measured using a total organic carbon analyzer (TOC-820, Sievers, CO, US) and TOC-TN analyzer (TOC-V CPH, Shimadzu, Japan), respectively. Sediment samples were taken from both Acorus and Typha wetlands. All the sediment samples were collected in triplicate from upper 1 cm layers, transported to the laboratory in an ice box, and then stored at −20 °C until the experimental processings took place. 2.2. Extraction of DNA and PCR DNA was extracted from 0.25 g of soil using the PowerSoil DNA isolation Kit (PowerSoil, Mobio laboratories Inc., CA, US), according to the provided instructions. The DNA extractions were performed in quadruplicate from each soil sample. The concentration of DNA was quantified using a Nano-drop ND-100 UV–vis spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA) at 260 nm. The nirS gene was amplified in a mastercycler (Eppendorf Mastercycler personnel, Germany) with the primer pair cd3aF (5′GT(C/G) AAC GT(C/G) AAG GA(A/G) AC(C/G) GG-3′) and R3cd (5′-GA (C/G) TTC GG(A/G) TG(C/G) GTC TTG A-3′). The PCR mixture included PCR premix (AccuPower™ PCR HotStart PreMix kit, Bioneer, Korea), 0.25 μM of each primer, and 30 ng of template DNA. The PCR cycling was performed by using an initial denaturation step of 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, elongation at 72 °C for 30 s, and a final extension at 72 °C for 10 min. All of the PCR products were analyzed on 1.5% (w/v) agarose gels and visualized after staining with ethidium bromide. 2.3. Real time PCR The real time PCR assay was performed on the Rotor-Gene 6000 (Corbett research, NSW, Australia) using the SYBR green detection

system with the primer pairs nirS2F (5′-TAC CAC CC(C/G) GA(A/G) CCG CGC GT-3′) and nirS3R (5′-TTC CT(C/G/T) CA(C/T) GAC GGC GGC3′). Real time PCR for nirS was performed in a volume of 20 μL and the reaction mixture contained 10 μL 2 × SensiMixPlus (Quantace, Norwood, MA), 0.25 μM of each primer, 1 μL of template DNA, and RNasefree water. The thermocycling steps of the real time PCR for nirS amplification included 10 min at 95 °C, 35 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. Standard curves were generated by plotting the threshold cycle values versus log10 of the gene copy numbers. The standards were prepared by using serially diluted plasmid DNA with 103 to 108 gene copies μL−1. The specificity of the amplified products was confirmed using both melting curve analysis and agarose gel electrophoresis. Two independent real time PCR assays were performed on each of the three replicate soil DNAs. A student t test was performed to examine significant differences in nirS gene copy numbers among samples.

2.4. Cloning and sequencing The PCR products were purified using the AccuPrep PCR purification kit (k-3034, Bioneer, Korea). The purified PCR products were cloned using the TOPcloner-TA-V2 vector (Enzynomics, Korea) following the manufacturer's instructions, and transformed into Escherichia coli DH5α competent cells (Enzynomics, Korea). Colonies were screened by Colony PCR with vector primers M13F (5′-GTA AAA CGA CGG CCA G-3′) and M13R (5′-CAG GAA ACA GCT ATG AC-3′). A single colony containing the recombinant plasmids was inoculated into 1 mL LB broth with ampicilin (60 μg/mL), and incubated at 37 °C for 14–16 h. The plasmid DNA was then extracted and purified using the Gene All quick plasmid kit (GeneAll Biotechnology, Korea) according to the manufacturer's instructions. Clones with the correct insert were sequenced using an ABI 3730xl automated DNA analyzer (Applied Biosystems, Foster City, CA) at Xenotech (Daejeon, Korea).

2.5. Phylogenetic and diversity analysis The DNA sequences were translated into amino acid sequences by using ExPASy Proteomics tools (http://www.expasy.ch/tools/dna.html). The amino acid sequences were compared against database sequences using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/blast). The protein sequences were aligned by using the ClustalX program (version 1.81) [21] available in the BioEdit software package [22], and the distance matrices were calculated by using the PHYLIP protdist program (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=protdist). The DOTUR program [20] was used to group the valid nirS sequences into operational taxonomic units (OTUs) with a 95% sequence similarity. The indices of diversity (Simpson D, Shannon-Weiner (H′), and Chao 1) and evenness were calculated by using DOTUR. The phylogenetic analyses of deduced amino acid sequences were conducted by using MEGA version 3.1 [23]. The tree was constructed from a matrix of pairwise genetic distances by using the neighbor-joining method with the

Table 1 Water characteristics for wastewater effluents and samples in the constructed wetlands. Temperature (°C)

pH

3 September, 2009 WWTP effluent Acorus Typha

24.7 24.2 27.4

7.0 6.8 6.9

17 November, 2009 WWTP effluent Acorus Typha

15.4 9.6 10.3

6.8 6.8 7.2

DOC (mg C/l)

NO− 2 (mg N/l)

NO− 3 (mg N/l)

TN (mg/l)

TN removal (%)

184 208 −15

14.9 ± 1.3 13.2 ± 0.1 10.4 ± 0.1

0.7 ± 0.1 0.2 ± 0.1 0.0 ± 0.0

32.0 ± 0.1 28.1 ± 0.2 0.0 ± 0.1

34.5 ± 0.8 27.3 ± 0.9 1.8 ± 0.2

73.8

270 256 −135

6.1 ± 0.9 5.4 ± 0.3 14.2 ± 1.0

0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

12.7 ± 0.2 7.3 ± 0.1 0.0 ± 0.0

13.3 ± 0.9 8.5 ± 0.8 1.3 ± 0.2

53.2

ORP (mV)

K. Chon et al. / Desalination 264 (2010) 201–205 Table 2 Diversity indices and predicted richness of nirS clones from Acorus and Typha wetland sediment samples, as estimated with the Simpson and Shannon-Weiner indices and Chao 1 richness estimators computed using DOTUR. Soil No. of No. of OTUs Simpson Shannon- Chao 1 sample clones (5% cut) index (1/D) Weiner index (H′) Sept.03.09 Acorus Typha Nov.17.09 Acorus Typha a

48 52 56 55

27 39 25 35

20.16 88.50 15.87 51.28

3.02 3.58 2.87 3.42

44.00 76.80 51.25 60.67

Ea

0.92 0.98 0.89 0.96

E, evenness calculated as E = H′/ln(number of OTUs).

Amino Poisson correction model. The consensus trees were calculated after bootstrapping with 1000 replicate trees. 2.6. Nucleotide sequence accession numbers The nucleotide sequences of nirS reported here have been deposited in GenBank under the accession numbers GU393027 to GU393237. 3. Results and discussion 3.1. Water characteristics of samples Table 1 represents the characteristics of the wastewater and wetlands effluent samples, in terms of temperature, pH, ORP, DOC, and nitrogen levels, during September and November of 2009. All the samples had neutral pH values. The concentration of DOC slightly decreased throughout the wetlands in the summer but increased twice as much in the Typha wetland as compared to the wastewater effluents in the winter, which was presumably resulted from the leaching of organics from the wetland sediments, under anoxic conditions exhibiting a relatively lower ORP value than it did in the summer. Most of the TN consisted of NO− 3 –N in wastewater effluents, as shown in Table 1. Nitrate level continuously decreased through the wetlands, and was almost completely removed in the Typha wetland. This result was in agreement with our previous study, which showed that nitrate was efficiently removed especially by the Typha wetland [17]. Removal efficiency of the TN was higher in September than in November, in the Typha wetland, indicating that the denitrification rate was affected by temperature. It was supposed that the diversity and abundance of denitrifiers were higher for the Typha wetland at higher temperatures. 3.2. Diversity of the nirS sequences The diversity of nirS in constructed wetland sediments was examined by using clone library analysis (Table 2). A total of 211 clones were sequenced in this study. NirS clones were obtained from the Acorus and Typha wetland sediment samples collected on Septmeber 3rd and November 17th, 2009. The diversity and richness indices of nirS were higher in the Typha wetland sediments collected in both September and November than in the Acorus wetland sediments. The Typha wetland sediment samples collected on September 3rd contained the highest diversity and richness. These results can be correlated with higher TN removal efficiency from the Typha wetland sample in the warmer season. Fig. 1. Phylogenetic tree constructed by the neighbor-joining method based on the deduced nirS amino acid sequences cloned from Acorus (A) and Typha (T) wetland sediment samples. Clones with more than 95% sequence similarity were considered to be the same operational taxonomic unit (OUT). Sequences with names staring with 9 and 11 represent the samples collected on September 3rd and November 17th, 2009, respectively. The total numbers of clones in each OTU are shown in parentheses. Bootstrap values greater than 50% (1000 replicates) are shown. Pyrobaculum aerophilum (NP_560850) was used as the out-group to root the phylogram. The scale bar represents one substitution per ten nucleotides.

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K. Chon et al. / Desalination 264 (2010) 201–205 Table 3 Relative abundance of nirS sequences obtained from cloned samples in every cluster defined from the phylogenetic analysis. Cluster

Relative abundance of nirS sequences from each clone library (%) Sept.03.09

A B C D E F G H

Fig. 1. (continued).

3.3. Phylogenetic analysis of nirS sequences The phylogenetic tree of deduced amino acids can be divided into eight (A to H) clusters (Fig. 1). A, B/G, D, and E were related to the nirS of Xanthomonadales, Burkholderiales, Rhodocyclales, and Hydrogenophilales bacteria, respectively. These bacteria have been reported to be dominant in wastewater treatment plants [24–27]. Especially, Burkholderiales and Rhodocyclales bacteria have been known to dominate in the denitrification process in wastewater treatment systems [25,27]. Meanwhile, the remaining clones in clusters C, F, and H were relatively less related to the nirS gene of cultivated denitrifying bacteria. Cluster A (36 clones) consisted of clones with similarities to a previous sequence of 82–97%, Rhodanobacter sp. (AB480490), being the closest cultivated denitrifiers. Cluster B (18 clones) showed 74– 86% similarity to nirS sequences from known denitrifying bacteria belonging to the orders Burkholderiales, such as Brachymonas denitrificans (DQ865925), Cupriavidus sp. (AB480486), Ralstonia metallidurans (NC_007973), and Comamonas denitrificans (DQ865926). Clones, included in cluster C (17 clones), were found to be 71–99% related to nirS sequences mostly retrieved from the constructed wetland sediments and were 60–88% identical to cultivated bacteria of Magnetospirillum (CU459003). The nirS clones in cluster D (37 clones) were related to nirS sequences from Thauera

No. of sequences

Nov.17.09

Acorus (9AS)

Typha (9TS)

Acorus (11AS)

Typha (11TS)

6 8 11 23 4 38 10

25 7 6 6 2 44 4 6

5 11 9 41

31 7 7

32 2

4 45 2 4

36 18 17 37 5 84 8 6

sp. (AY07826, AM230892) and Dechloromonas sp. (AM230913) belonging to the order Rhodocyclales at 71–94% identity. Members of cluster E (5 clones) were 84–99% related to environmental clones and exhibited 79–82% similarity with known bacteria Thiobacillus denitrificans (NC_007404). Cluster F (84 clones) was composed of clones sharing 73–100% similarity with environmental sequences obtained from constructed wetland [16], arable soil [14], rice field soil [18], and forest soil [28]. No clone in cluster F had a similarity greater than 80% to previously identified species. These results indicate that denitrifiers with previously uncharacterized nirS genes were dominantly present in the constructed wetland soil. Members of cluster G (8 clones) were 80–94% related to sequences obtained from activated sludge [29] and showed 78–85% similarity with denitrifier isolates, Acidovorax sp. (NC_008782). Cluster H (6 clones) consisted of nirS sequences with the lowest identities to environmental clones. Relative abundances of clones grouped in clusters A–H varied according to the sampling date and different wetland types (Table 3). Most clones from Acorus wetland sample belonged to clusters D and F. The nirS clones, belonging to clusters A and F, existed dominantly in the Typha wetland in both seasons. The result suggests that bacteria harboring not-yet-characterized nirS relatively dominate in the Acorus and Typha wetlands in both seasons. The nirS clones in cluster H and G/E were not detected in 9AS and 11AS samples, respectively. The nirS clones, belonging to cluster D, were not represented in 11TS samples. The Acorus wetland contained relatively higher numbers of clones belonging to cluster D, which failed to thrive in the Typha wetland. In contrast, relatively higher numbers of clones belonging to cluster A were detected in the Typha wetland, which were less represented in the Acorus wetland. This result suggests that nirS related to Rhodocyclales and Xanthomonadales bacteria were more abundant than others in the Acorus and Typha wetlands, respectively. 3.4. Abundance of nirS Copy numbers of nirS from both Acorus and Typha wetlands in the summer were significantly higher than those of the nirS gene in the winter (Table 4). In addition, nirS copy numbers of Acorus wetlands were approximately 10 times greater than those of Typha wetlands in both seasons. This result also indicates a similar trend in our previous study [19]. However, this does not necessarily indicate that

Table 4 Copy numbers of nirS (× 108copies/g soil) in Acorus and Typha wetland sediment samples determined by real time PCR. nirS

Sept.03.09

Nov.17.09

Probability

Acrous Typha Probability

13.2 ± 2.0 1.8 ± 0.2 0.0014

8.6 ± 1.1 0.7 ± 0.1 0.0006

0.027 0.002

K. Chon et al. / Desalination 264 (2010) 201–205

denitrification activity in the Acorus wetlands is higher than in the Typha wetlands. Thus, mRNA analysis is necessary to enhance our understanding of metabolic activity of nirS genes in each wetland. 4. Conclusions The diversity and abundance of nirS genes were investigated, using clone library analysis in constructed wetlands, to demonstrate nitrogen removal efficiency in a constructed wetland fed with a wastewater effluent, containing nitrate. Similarly to previous studies, most clones were less closely related to the nirS gene of known denitrifiers in the Acorus and Typha wetlands. Some clones harboring nirS were identified to be related to those of known Xanthomonadales and Rhodocyclales bacteria, which were mainly found in the Typha and Acorus wetlands, respectively. These results indicate that the previously unidentified denitirifers, Xanthomonadales, and Rhodocyclales bacteria, may play an important role in denitrification in the tested constructed wetland. The Typha wetland had higher diversity and richness indices of nirS (Simpson D, Shannon-Weiner (H′), and Chao 1), regardless of seasons, compared to the Acorus wetland, showing good agreement with higher nitrogen removal efficiency in the Typha wetland. The nirS genes were also found to be more abundant in the summer (Acorus: 13.2 ± 2.0, Typha: 1.8 ± 0.2× 108 copies/g soil) than in the winter (Acorus: 8.6 ± 1.1, Typha: 0.7 ± 0.1 × 108 copies/g soil) in constructed wetlands. These results suggest that the diversity and abundance of nirS respond to environmental changes, such as temperature and nitrogen load. Acknowledgements We thank Ms. Ryu Jiyoung (GIST, Korea) and Mr. Lee Taekwon (Yonsei Universty, Korea) for their comments on the data analysis. We thank Prof. In S. Kim, Prof. Kyoung-Woong Kim, and Prof. Hor-Gil Hur for allowing us to use their real time PCR, PCR thermal cycler, and Nano-drop spectrophotometer instruments, respectively. This research was supported by a grant from the National Research Laboratory Program by the Korea Science and Engineering Foundation (NOM Lab: R0A-2007-000-20055-0). Lastly, we hope to give our best and warmest appreciation and respects to Prof. Miriam Balaban, for her invaluable efforts and works. References [1] R.H. Kadlec, R.L. Knight, Treatment Wetlands, Lewis Publishers, Boca Raton, NY, 1996, p. 71. [2] B. Brix, C.A. Arias, The use of vertical flow constructed wetlands for on-site treatment of domestic wastewater: new Danish guidelines, Ecol. Eng. 25 (2005) 491–500. [3] J. Vymazal, Horizontal sub-surface flow and hybrid constructed wetlands systems for wastewater treatment, Ecol. Eng. 25 (2005) 478–490. [4] M.A. Maine, N. Suñe, H. Hadad, G. Sánchez, C. Bonetto, Nutrient and metal removal in a constructed wetland for wastewater treatment from a metallurgic industry, Ecol. Eng. 26 (2006) 341–347. [5] Z. Song, Z. Zheng, J. Li, Z. Sun, Z. Han, W. Wang, M. Xu, Seasonal and annual performance of a full-scale constructed wetland system for sewage treatment in China, Ecol. Eng. 26 (2006) 272–282.

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