Elevated Salinity Inhibits Nitrogen Removal by Changing the Microbial Community Composition in Constructed Wetlands During the Cold Season

Marine and Freshwater Research, 2018, 69, 802–810 © CSIRO 2018 http://dx.doi.org/10.1071/MF17171

Supplementary material

Elevated salinity inhibits nitrogen removal by changing the microbial community composition in constructed wetlands during the cold season

Yajun QiaoA,B,1, Penghe WangA,C,1, Wenjuan ZhangA, Guangfang SunA, Dehua ZhaoA,B,D, Nasreen JeelaniA,B,

Xin LengA,B,D, and Shuqing AnA,B

AInstitute of Wetland Ecology, School of Life Science, Nanjing University,

Xianlin Avenue 163, Nanjing, 210046, P.R. China.

BNanjing University Ecology Research Institute of Changshu, Huanhu Road 1,

Changshu, 215500, P.R. China.

CMCC Huatian Engineering and Technology Corporation, Fuchunjiangdong Street 18,

Nanjing, 210019, P.R. China.

DCorresponding authors. Email: [email protected]; [email protected]

Page 1 of 7 Marine and Freshwater Research © CSIRO 2018 http://dx.doi.org/10.1071/MF17171

Fig. S1. The subsurface flow constructed wetlands (SSF-CWs) and microorganism sampling points. The substrates include three layers: a bottom layer (gravel; porosity, 0.55; diameter,

30–50 mm; thickness, 550 mm), middle layer (gravel; porosity, 0.45; diameter, 10–20 mm; thickness, 100 mm), and top layer (sand; diameter, 1–2 mm; thickness, 100 mm).

Page 2 of 7 Marine and Freshwater Research © CSIRO 2018 http://dx.doi.org/10.1071/MF17171

Fig. S2. The daily water temperature of the influent during the preprocessing period and experimental period. A Temperature and Light Data Logger (HOBO UA-002–08; Onset, Cape

Cod, MA, USA) was used to record the water temperature.

Page 3 of 7 Marine and Freshwater Research © CSIRO 2018 http://dx.doi.org/10.1071/MF17171

Table S1. The detailed information for 16S rRNA gene qPCR and Illumina MiSeq sequencing analysis in this study Information qPCR Illumina MiSeq sequencing Analysis system Illumina-Eco real-time PCR system (Illumina, San Diego, Illumina MisEqn 2500 sequencing platform (Illumina, San Diego, CA, USA) CA, USA) Primer pair 690F (5-TGTGTAGCGGTGAAATGCG-3) 829R (5- 341F (5ʹ-CCTAYGGGRBGCASCAG-3ʹ) 785R (5ʹ-GACTACHVGGGTATCTAATCC-3ʹ) CATCGTTTACGGCGTGGAC-3) Reaction mixture 5.0 μL of SYBR Premix Ex Taq II (Takara, Otsu, Japan), 1.0 25 μL of reaction mixture (including 10 ng of template, 0.5 μL of forward primer, 0.5 μL of μL of template DNA (diluted 100-fold), 0.5 μL of forward reverse primer) and 0.5 μL of reverse primer (10 μM), 3.0 μL of RNase-free water PCR program 30 s at 94°C, 40 cycles of 5 s at 95°C, 30 s at 55–60°C, and 3 min at 94°C, 30 cycles of 10s at 94°C, 15s at 55°C, and 72°C for 30 s, and a final 30 s at 72°C incubation at 72°C for 7 min PCR product / Agencourt AMPure beads (Beckman Coulter, Inc., Fullerton, CA, USA) purification Libraries / NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs Inc., Boston, construction MA, USA)

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Table S2. Alpha diversity analysis at 3% dissimilarity based on the 16S rRNA gene Miseq sequencing analysis

The richness estimators (Chao1), diversity indices (Simpson), evenness indices (Shannon-even), and coverage (Good’s coverage index) were calculated using Mothur

program.CWP, Planted SSF-CWs fed with normal wastewater; CWP+, Planted SSF-CWs fed with saline wastewater; CWU, Unplanted SSF-CWs fed with normal

wastewater; CWU+, Unplanted SSF-CWs fed with saline wastewater. Microbial samples were collected on 10 January 2016

Samples OTUs Chao1 Simpson Shannon- Coverage (×104) even CWP 18896 62781 9.28 0.914 0.763 CWP+ 10930 41143 7.63 0.857 0.868 CWU 13288 42601 4.59 0.889 0.862 CWU+ 15339 37275 3.48 0.909 0.854

Table S3. The main genera (relative abundance >0.50%) with the addition of three nitrifying bacteria (Nitrosomonas, Nitrosospira and Nitrospira) in the four systems

Primary genera (relative abundance >2.00%) in each sample are given in bold. CWP, Planted SSF-CWs fed with normal wastewater; CWP+, Planted SSF-CWs fed

with saline wastewater; CWU, Unplanted SSF-CWs fed with normal wastewater; CWU+, Unplanted SSF-CWs fed with saline wastewater. Microbial samples were

collected on 10 January 2016

Phylum Class Order Family Genus Reads numbers Relative abundances (%) CWP CWP+ CWU CWU+ CWP CWP+ CWU CWU+ Proteobacteria β-proteobacteria Burkholderiales Burkholderiaceae Polynucleobacter 203 24 15 0 0.62 0.05 0.04 0 Proteobacteria β-proteobacteria Burkholderiales Comamonadaceae Albidiferax 1538 15 443 39 4.69 0.03 1.09 0.08 Proteobacteria β-proteobacteria Burkholderiales Comamonadaceae Hydrogenophaga 197 0 25 3 0.6 0 0.06 0.01 Proteobacteria β-proteobacteria Burkholderiales Comamonadaceae Polaromonas 320 5 30 4 0.98 0.01 0.07 0.01 Proteobacteria β-proteobacteria Burkholderiales Comamonadaceae Simplicispira 222 2 9 1 0.68 0 0.02 0 Proteobacteria β-proteobacteria Methylophilales Methylophilaceae Methylophilus 317 3 4 6 0.97 0.01 0.01 0.01 Proteobacteria β-proteobacteria Neisseriales Neisseriaceae Chitinibacter 245 7 14 9 0.75 0.01 0.03 0.02 Proteobacteria β-proteobacteria Neisseriales Neisseriaceae Deefgea 1260 13 80 14 3.85 0.03 0.2 0.03 Proteobacteria β-proteobacteria Neisseriales Neisseriaceae Neisseria 8 315 200 306 0.02 0.63 0.49 0.59 Proteobacteria β-proteobacteria Rhodocyclales Rhodocyclaceae Azospira 183 3 1 0 0.56 0.01 0 0 Proteobacteria β-proteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas 365 3 19 2 1.11 0.01 0.05 0 Proteobacteria β-proteobacteria Rhodocyclales Rhodocyclaceae Sulfuritalea 306 2 11 0 0.93 0 0.03 0 Proteobacteria γ-proteobacteria Aeromonadales Aeromonadaceae Aeromonas 798 29 31 10 2.44 0.06 0.08 0.02 Proteobacteria γ-proteobacteria Aeromonadales Aeromonadaceae Tolumonas 892 7 145 16 2.72 0.01 0.36 0.03 Proteobacteria γ-proteobacteria Alteromonadales Alteromonadaceae Haliea 372 1 0 0 1.14 0 0 0 Proteobacteria γ-proteobacteria Alteromonadales Shewanellaceae Shewanella 528 13 34 1 1.61 0.03 0.08 0 Proteobacteria γ-proteobacteria Chromatiales Chromatiaceae Rheinheimera 238 2 8 5 0.73 0 0.02 0.01 Proteobacteria γ-proteobacteria Enterobacteriales Enterobacteriaceae Serratia 836 30348 24983 31742 2.55 60.98 61.5 61.39 Proteobacteria γ-proteobacteria Oceanospirillales Halomonadaceae Halomonas 1978 11028 6766 11301 6.04 22.16 16.66 21.86

Phylum Class Order Family Genus Reads numbers Relative abundances (%) CWP CWP+ CWU CWU+ CWP CWP+ CWU CWU+ Proteobacteria γ-proteobacteria Pseudomonadales Moraxellaceae Enhydrobacter 0 495 466 573 0 0.99 1.15 1.11 Proteobacteria γ-proteobacteria Pseudomonadales Pseudomonadaceae Cellvibrio 212 5 15 1 0.65 0.01 0.04 0 Proteobacteria γ-proteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas 613 434 381 547 1.87 0.87 0.94 1.06 Proteobacteria δ-proteobacteria Bdellovibrionales Bacteriovoracaceae Bacteriovorax 611 8 106 12 1.86 0.02 0.26 0.02 Proteobacteria δ-proteobacteria Bdellovibrionales Bacteriovoracaceae Peredibacter 505 5 51 2 1.54 0.01 0.13 0 Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae Corynebacterium 11 577 738 663 0.03 1.16 1.82 1.28 Actinobacteria Actinobacteria Actinomycetales Dietziaceae Dietzia 3 160 237 282 0.01 0.32 0.58 0.55 Actinobacteria Actinobacteria Actinomycetales Propionibacteriaceae Propionibacterium 13 565 797 1032 0.04 1.14 1.96 2 Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae Paludibacter 253 2 14 1 0.77 0 0.03 0 Bacteroidetes Flavobacteria Flavobacteriales Cryomorphaceae Algoriphagus 415 20 35 2 1.27 0.04 0.09 0 Bacteroidetes Flavobacteria Flavobacteriales Cryomorphaceae Fluviicola 275 1 21 1 0.84 0 0.05 0 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Flavobacterium 6890 111 234 84 21.03 0.22 0.58 0.16 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Luteolibacter 611 0 15 1 1.86 0 0.04 0 Bacteroidetes uncertain uncertain uncertain Ohtaekwangia 2574 14 130 10 7.86 0.03 0.32 0.02 Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus 7 875 560 793 0.02 1.76 1.38 1.53 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus 5 469 342 443 0.02 0.94 0.84 0.86 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus 10 890 526 891 0.03 1.79 1.29 1.72 Flavobacteria Sphingobacteria Sphingobacteriales Crenotrichaceae Terrimonas 171 7 7 0 0.52 0.01 0.02 0 Verrucomicrobia Verrucomicrobiae Opitutales Opitutaceae Opitutus 330 3 39 3 1.01 0.01 0.1 0.01 Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobiaceae Prosthecobacter 343 3 29 4 1.05 0.01 0.07 0.01 OD1 uncertain uncertain uncertain uncertain 774 0 97 1 2.36 0 0.24 0 Subdivision3 uncertain uncertain uncertain uncertain 657 1 29 5 2.01 0 0.07 0.01 Proteobacteria β-proteobacteria Nitrosomonadales Nitrosomonadaceae Nitrosomonas 3 0 0 0 0.0092 0 0 0 Proteobacteria β-proteobacteria Nitrosomonadales Nitrosomonadaceae Nitrosospira 2 0 0 1 0.0061 0 0 0.0019 Nitrospirae Nitrospira Nitrospirales Nitrospiraceae Nitrospira 28 3 1 0 0.0855 0.006 0.0025 0

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