Ecotoxicology DOI 10.1007/s10646-014-1333-4

Distribution patterns of ammonia-oxidizing and anammox bacteria in the freshwater marsh of Honghe wetland in Northeast China

Kwok-Ho Lee • Yong-Feng Wang • Guo-Xia Zhang • Ji-Dong Gu

Accepted: 9 August 2014 Ó Springer Science+Business Media New York 2014

Abstract Community characteristics of aerobic ammonia- suggesting that the utilization of hzo biomarker for detecting oxidizing bacteria (AOB) and anaerobic ammonium-oxi- anammox bacteria in freshwater marsh might have serious dizing (anammox) bacteria in Honghe freshwater marsh, a limitations. Results with 16S rRNA gene showed that Ramsar-designated wetland in Northeast China, were ana- Candidatus Kuenenia was detected in only the Experimental lyzed in this study. Samples were collected from surface and Zone, whereas Ca. Scalindua including different lineages low layers of sediments in the Experimental, Buffer, and was observed in both the Buffer and Experimental Zones but Core Zones in the reserve. Community structures of AOB not the Core Zone. These results indicated that both AOB were investigated using both 16S rRNA and amoA (encod- and anammox bacteria have specific distribution patterns in ing for the a-subunit of the ammonia monooxygenase) the ecosystem corresponding to the extent of anthropogenic genes. Majority of both 16S rRNA and amoA gene-PCR impact. amplified sequences obtained from the samples in the three zones affiliated with Nitrosospira, which agreed with other Keywords Nitrification Anammox Wetland wetland studies. A relatively high richness of b-AOB amoA Anthropogenic pollution Hydrazine oxidoreductase gene detected in the freshwater marsh might suggest mini- mal external pressure was experienced, providing a suitable habitat for b-AOB communities. Anammox bacteria com- Introduction munities were assessed using both 16S rRNA and hzo (encoding for hydrazine oxidoreductase) genes. However, Wetlands are an important ecosystem on earth, supporting PCR amplification of the hzo gene in all samples failed, a high diversity of a wide range of organisms and in turn serve a important function in food production and nutrients Kwok-Ho Lee and Yong-Feng Wang are contributed to this work cycling (Gopal and Ghosh 2008; Nagelkerken et al. 2008). equally. They act as an important breeding site for endangered wildlife species, such as protected and threatened flora and K.-H. Lee J.-D. Gu (&) avifauna (Nagelkerken et al. 2008). They also play a vital Laboratory of Environmental Microbiology and Toxicology, role in cycling of different nutrients and mitigation of School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, People’s Republic of China climate change through the marsh ecosystem (Zhou et al. e-mail: [email protected] 2009). Hence the ecological functions of wetland have important implications to both the terrestrial and aquatic Y.-F. Wang environments nearby and beyond. The biogeochemical Laboratory of Microbial Ecology, Guangdong Academy of Forestry, No. 233, Guangshan 1st Road, Guangzhou, People’s nitrogen cycle in marsh is a unique characteristics, which Republic of China plays a critical role in decomposition and pollution control (Birol et al. 2006). G.-X. Zhang With the high microbial biodiversity, microbial nitrifi- Department of Environmental Health, School of Public Health and Tropical Medicine, Southern Medical University cation and denitrification facilitate the nitrogen removal in Guangzhou, Guangdong 510515, People’s Republic of China the marsh (Zhu et al. 2010). Denitrification takes place 123 Kwok-Ho Lee et al. under anoxic environment in which nitrate is reduced 2013), coastal wetlands (Wang and Gu 2014; Wang et al. stepwise to dinitrogen gas (N2) as the end-product released 2014a), freshwater lake (Schubert et al. 2006), freshwater into the atmosphere by heterotrophic organisms with high river (Sun et al. 2014), and various soils types (Humbert diversity of bacteria (You et al. 2009). Nitrification is et al. 2009; Humbert et al. 2012; Wang et al. 2013a). carried out by two steps: aerobic process in which However, only a few reports are available on the anammox ammonia is oxidized to nitrite via hydroxylamine, and then bacteria in natural saline and constructed wetlands (Erler nitrite further conversion to nitrate. The first process is et al. 2008; Humbert et al. 2009). For our knowledge, carried out by both ammonia-oxidizing bacteria (AOB) and anammox bacteria have not been reported in freshwater more recently archaea (AOA) (Cao et al. 2013;Ko¨nneke marshes. et al. 2005). The intermediate nitrite is seldom accumulated In this study, we reported the diversity of AOB and in natural environments due to the rate-limiting step of anammox bacteria in Honghe National Freshwater Marsh ammonia oxidation process. The nitrite produced by the sediments using molecular analysis approaches to amplify first nitrification step would be utilized by the nitrite-oxi- both 16S rRNA gene (i.e. 16S rDNA) and functional genes dizing bacteria (NOB) in later process of the nitrification of both microbial groups. This research provides important (Kowalchuk and Stephen 2001). information whether freshwater marsh ecosystem is a It was believed nitrification could only occur under aer- suitable environment for AOB and anammox bacteria. obic condition as ammonia remains in inert ionic state under anoxic conditions until the discovery and confirmation of anaerobic ammonium oxidation (anammox) from a study in Materials and methods which nitrogen gas was evolved from anaerobic fluidized bed bioreactors of a wastewater treatment plant (Mulder et al. Site description and sample collections 1995). Coupling of nitrification and denitrification in the marsh ecosystem is mainly confined in saturated sediment Honghe National Freshwater Marsh is listed as a Ramsar and other submerged subsurface including the rhizosphere of protected wetland and belongs to one of the typical natural vegetation (Ruiz-Rueda et al. 2009). AOB was first isolated inland freshwater marshes in Sanjiang Plain (Fig. 1), which 100 years ago (Frankland and Frankland 1890), but up to is an extensive area between three big rivers: the Heilong now, the phylogeny of AOB includes family Nitrosomo- River, the Songhua River and the Wusuli River in the nadaceae in the b- with the two genera, northeast of China (Zhou et al. 2009). The wetland is and Nitrosospira (Purkhold et al. 2000; located at the boundary area of Tongjiang City and Fuyuan Purkhold et al. 2003) and c-proteobacteria formed from a County, and within the Jiansanjiang Sub-bureau of General single genus Nitrosococcus which is strictly aerobic and only Bureau of Heilongjiang State Farms and Land Reclamation detected in marine environments (Kowalchuk and Stephen Administrative region with an area of around 21,835 hm2 2001). The b-AOB were observed in diverse habitats, (Liu 2009). Honghe wetland has been divided into three including bays (O’Mullan and Ward 2005), coastal wetlands management parts, namely Experimental Zone (E) (2,000 (Cao et al. 2012; Wang et al. 2013c; Wang and Gu 2013; hm2), Buffer Zone (B) (12,835 hm2) and Core Zone Zheng et al. 2013), saline lake (Jiang et al. 2009), freshwater (C) (7,000 hm2). The Core Zone is the main feeding ground marsh (Laanbroek and Speksnijder 2008), saline wetland and breeding habitat for the endangered birds species like (Dorador et al. 2008), paddy soils (Wang et al. 2013b; Wang Glycine soja, Acanthlpanax senticosus, Astragalus mem- et al. 2014b), volcanic soil (Herna´ndez et al. 2014) and ter- branaceus, Phellodendron amurense, Juglans mandshurica restrial soils (Jiang et al. 2014; Nicol et al. 2008; Norman and and Fraxinus mandshurica. Barrett 2014; Sher et al. 2013; Strauss et al. 2014; Zhou et al. In this study, surface layer (1–2 cm) and low layer 2014). (20–22 cm) of sediment samples from each of the three zones The bacteria responsible for the anammox process were collected in August 2008 and immediately transferred belong to the phylum Planctomycetes. Five genera of back to the laboratory in an ice packed cooler. Each sample anammox bacteria have been reported so far: Candidatus was separated into three equal parts from which one part was Brocadia (Kuenen and Jetten 2001), Ca. Kuenenia (Schmid for DNA extraction with two replicates per sample, second et al. 2000), Ca. Scalindua (Kuypers et al. 2003), Ca. part was for chemical analysis, and the third part was stored Anammoxoglobus (Kartal et al. 2007) and Ca. Jettenia under -80 °C for further investigation if needed. (Quan et al. 2008). Anammox bacteria have a wide dis- tribution in various oxygen-limited marine environments Analysis of chemical characteristics (Stevens and Ulloa 2008), marine surface water (Rich et al. 2008), marine sediment and water (Li et al. 2010; Shao Concentrations of ammonium-N, nitrate–N, and nitrite-N et al. 2014; Song et al. 2014; van de Vossenberg et al. were measured by extracting 100 g of sediments in 2.0 M 123 Distribution patterns of ammonia-oxidizing bacteria

Fig. 1 Sampling sites in the Honghe National Nature Reserve at Sanjiang Plain, Northeast China

Table 1 A list of primer combinations and corresponding PCR protocols used in this study Target group Primer Primer sequences (50-30) Amplicon Thermal profile for PCR Reference length (bp)

Bacterial 16S 27F GTGCTGCAGAGAGTTTGATCCTGGCTCAG 1,365 95 °C for 5 min followed by 34 (Rochelle rDNA gene 1392R CACGGATCCACGGGCGGTGTGTRC cycles of 45 s at 94 °C, 1 min at et al. 1995) 55 °C, 1 min at 72 °C AOB 16S NitA CTTAAGTGGGGAATAACGCATCG 1,055 95 °C for 2 min followed by 30 (Voytek and rDNA gene NitB TTACGTGTGAAGCCCTACCCA cycles of 30 s at 95 °C, 30 s at Ward 1995) 57 °C, 3 min at 73 °C AOB amoA amoA-1F GGGGTTTCTACTGGTGGT 491 94 °C for 3 min followed by 37 (Okano et al. gene amoA-2R CCCCTCKGSAAAGCCTTCTTC cycles of 30 s at 94 °C, 30 s at 2004) 55 °C, 45 s at 72 °C Anammox Amx368F TTCGCAATGCCCGAAAGG 452 94 °C for 4 min followed by 36 (Schmid et al. 16S rDNA Amx820R AAAACCCCTCTACTTAGTGCCC cycles of 45 s at 95 °C, 50 s at 2003) gene 59 °C, 1 min at 72 °C Plantomycete Pla46F GGATTAGGCATGCAAGTC 991 94 °C for 4 min followed by 30 (Neef et al. 16S rDNA cycles of 45 s at 95 °C, 50 s at 1998) gene 1037R CGACAAGGAATTTCGCTAC59 °C, 3 min at 72 °C (Ludwig et al. 1992) Anammox hzocl1F1 TGYAAGACYTGYCAYTGG 470 94 °C for 5 min followed by 34 (Schmid et al. hzo gene hzocl1R2 ACTCCAGATRTGCTGACC cycles of 1 min at 94 °C, 1 min 2008) at 50 °C, 90 s at 72 °C

KCl (Zuber et al. 2005) and the extracts were analyzed by DNA extraction and PCR amplification an auto-analyzer (QuikChem, Milwaukee, WI) using standard flow injection analysis (FIA) technique according Total genomic DNA was extracted from 150 to 250 mg of to the standard extraction procedures. sediments using SoilMasterTM DNA Extraction Kit,

123 Kwok-Ho Lee et al. according to the manufacturer’s protocol (Epicentre Bio- BLAST (http://www.ncbi.nih.gov) was used to find the technologies, Madison, WI). The DNA extracts were stored closest related 16S rDNA and amoA sequences in the at -20 °C before further use as templates for subsequent GenBank. The multiple alignments of partial 16S rDNA PCR amplification reactions. and amoA sequences were accomplished and neighbor- The following PCR primer sets specific for anammox joining phylogenetic trees with bootstrap values based on bacteria were: (1) Amx368F/Amx820R (16S rRNA gene) 1,000 replications (Felsenstein 1985) were produced using (Schmid et al. 2003); (2) hzocl1F1/hzocl1R2 (hzo gene: MEGA program. The sequence similarity of the screen encoding for hydrazine oxidoreductase) (Schmid et al. clones were performed using the closest relative in BLAST 2008); for b-AOB: (1) NitA/NitB (16S rRNA gene) which (GenBank) for amoA and 16S rDNA sequences and Ez- was suggested being relatively more specific than other AOB Taxon server by Chun et al. (2007) for 16S rDNA 16S rRNA gene primers (Dorador et al. 2008); (2) amoA-1F/ sequences and calculation by BioEdit software (To Hall, amoA-2R (amoA gene: encoding for the alpha-subunit of the North Carolina State University, NC). ammonia monooxygenase) (Okano et al. 2004) were selec- ted to amplify the corresponding genes. The details of Statistical analysis primers targeting different genes and corresponding PCR thermal profile used in this study are listed in Table 1. The DOTUR (Distance-Based OTU and Richness) program Nested PCR protocols were applied in amplifying both was employed to compare diversity for gene sequences from the anammox bacteria and b-AOB 16S rRNA gene (i.e. each sample (Schloss and Handelsman 2005). Operational 16S rDNA) to increase the anammox and b-AOB detection taxonomic units (OTU) for community analysis were defined limits reported (Humbert et al. 2009). The anammox and b- at a 3 % cutoff of nucleotide sequence variations in detecting AOB 16S rDNA were amplified with the Planctomycetes anammox bacteria and b-AOB as determined using the fur- 16S rDNA and 23S rDNA primers Pla46F/1037R (Table 1) thest neighbor algorithm in DOTUR. DOTUR was also used (Dale et al. 2009) and bacteria universal primers 27F/ to generate diversity analyses such as coverage, Chao1, 1392R (Table 1) (Dymock et al. 1996) using modified PCR Simpson index (D) and Shannon index (H) for each site. The mixture contents: GoTaqÒ DNA Polymerase (Promega, H index and the D index were calculated as the diversity Madison, WI) in a 25 ll volume containing 5 llof59 indices by the program (Schloss and Handelsman 2005). Ò colorless GoTaq Flexi Buffer, 12.5 mM MgCl2, 5 mM of Another recently developed and more advanced community each deoxyribonucleoside triphosphate, 0.5 lM of each analysis program MOTHUR was employed to conduct the primer, 1.25 U GoTaqÒ DNA Polymerase and 2 llof UniFrac Environment Clustering Analysis (Schloss et al. purified DNA as template (10–100 ng). The compositions 2009). The estimated coverage of the constructed 16S rDNA of bacterial amoA PCR 25 ll reaction mixture was iden- of anammox bacteria and b-AOB; bacterial amoA gene tical as that of 16S rDNA amplification, except using dif- libraries were calculated using the formula of C = [1-(n1/ ferent primer pairs whereas the PCR buffer concentration N)] 9 100, where n1 represents the number of OTUs in 25 ll hzo PCR mixture was reduced by half with other detected in one clone library and N strands for the total contents remained unchanged. number of clones in that particular library. This coverage estimates the probability that all the unique sequences Clone library construction and phylogenetic analysis present in a given sample were represented at least once in the library (Dang et al. 2008). Purified DNA fragments from the corresponding PCR amplifications were ligated into a pMD18-T vector (TaKa- Nucleotide sequence accession numbers Ra, Japan) and transformed into Escherichia coli DH5a cells. The library clones were screened directly by PCR for All partial 16S rRNA gene sequences of both anammox the presence of inserts using M13F and M13R (Table 1). The bacteria and b-AOB and amoA gene sequences of b-AOB positive clones from each library were randomly selected obtained in this study have been deposited in GenBank with and the PCR products were purified using a PCR Purification accession numbers HM537468 to HM537745, respectively. Kit (Qiagen, USA). The PCR products were sequenced at Genome Research Center of The University of Hong Kong. DNA sequences were examined and edited using Bio- Results Edit (Tom Hall, North Carolina State University, NC) and MEGA, version 5.0 (Tamura et al. 2011). Sequence chi- Chemical assay of sediments mera test from MOTHUR, which was a recently developed community analysis program, was performed for the chi- Detailed information about the chemical characteristics of meras check and detection (Schloss et al. 2009). NCBI sediments and the site locations are listed in Table 2. 123 Distribution patterns of ammonia-oxidizing bacteria

Table 2 Chemical parameters of the sediments in different sampling zones in the present study

? - - Zone Sample name Latitude (N) Longitude (E) Depth NH4 NO2 NO3 (cm) (lg/g DW) (lg/g DW) (lg/g DW)

Buffer S6 N47049024.100 E133040023.900 5 59.94 0.56 53.38 S7 N47049024.100 E133040023.900 15 32.61 0.24 6.54 Experimental S8 N47044050.000 E133037020.700 5 37.70 0.33 23.83 S9 N47044050.000 E133037020.700 15 15.19 0.22 3.03 Core HE-Top N47049045.000 E133041035.100 5 25.86 1.97 \0.1 HE-Bottom N47049045.000 E133041035.100 30 15.55 1.21 \0.1

Table 3 PCR detection of b-AOB and anammox bacteria in the Honghe National Natural Wetland Reserve Zone Sample name AOB 16S rDNA AOB amoA Anammox 16S rDNA Related clones Confirmeda Related clones Confirmedb Related clones Confirmedc

Buffer S6 20/24 ? 35/35 ? 30/33 ? S7 0/21 - N/A ND 24/33 ? Experimental S8 25/25 ? 20/20 ? 11/25 ? S9 14/24 ? 18/18 ? 26/30 ? Core HE-Top 6/27 ? N/A ND 0/33 - HE-Bottom 21/25 ? 17/17 ? 0/48 - a ? represent retrieved 16S rDNA sequences fall into AOB cluster b ? represent retrieved amoA sequences fall into AOB cluster c ? represent retrieved 16S rDNA sequences fall into anammox cluster - represent not confirmed ND not determined (negative PCR amplification)

Concentrations of inorganic nitrogen species including was chosen based on Junier et al. (2008). The primers ammonia, nitrite, and nitrate detected in the surface layer of NitA/NitB could detect the highest frequency of AOB the sediments in the Core Zone, Buffer Zone and Experi- including sequences more than other examined primer mental Zone were higher than that of low layer in all samples. combinations. In the application of nested PCR approach to The nitrite concentrations in the Core Zone was about 2–9 the AOB specific 16S rDNA PCR, none of the 16S rDNA times higher than that of the other two zones but both nitrate gene PCR sequences in sample 7 were related to any b- and ammonium levels in the Core Zone were the lowest AOB like sequences, neither Nitrosospira-like nor Nitros- among the three zones. Interestingly, the concentrations of omonas-like 16S rDNA sequences. The result suggested nitrite showed an increasing trend across the three zones that bacterial amoA gene amplification might be needed to where Experimental Zone, the outermost of the marsh, had prove the absence of AOB communities in sample 7, in the lowest nitrite level whereas that of Core Zone, the center particular. of the marsh, had the highest. But both layers of sediments in For AOB amoA gene, the PCR amplifications of sample the Buffer Zone showed the highest ammonium and nitrate 7 and HE-TOP were failed and only the remaining four levels among all samples. Ammonium concentrations across samples were amplified successfully (Table 3). The failure the three zones showed a significant positive correlation with of amplifying positive PCR products was not due to the that of the nitrate levels (Pearson moment correlation: quality of DNA as successful amplifications were detected r = 0.94, p \ 0.06, n = 6). Nitrate concentration in both for anammox bacteria and b-AOB 16S rDNA PCR of both surface and low layers of sediments in the Core Zone was samples, but it might be due to the low abundance of the below the detection limit (0.1 lg/g DW). amoA genes in the sediment samples. For 16S rDNA of anammox bacteria, a total of 202 General information of PCR assay on anammox selected clones among the six anammox bacteria clone bacteria and AOB libraries were sequenced. However, only 92 of the 202 sequences were related to anammox bacterial 16S rDNA The results of PCR amplification assays are shown in sequences, which included both the surface and low layers Table 3. For 16S rDNA of AOB, the primer set NitA/NitB of the sediments from Buffer and Experimental Zones 123 Kwok-Ho Lee et al.

Table 4 Diversity and Zones Buffer Experimental Core predicted richness of AOB 16S rDNA and amoA genes, and Samples S6 S7 S8 S9 HE-Top HE-Bottom anammox bacterial 16S rDNA AOB (16S rDNA) gene sequences recovered from the Honghe National Natural No. of clones 20 – 25 24 6 21 Wetland Reserve No. of OTUs (3 %) 2 – 1 1 2 1 Coverage (%) 90.00 – 96.00 95.83 66.67 95.24 Shannon Index 0.20 – 0.00 0.00 0.45 0.00 Simpson Index 0.90 – 1.00 1.00 0.67 1.00 Chao1 (3 %) 2.00 – 1.00 1.00 2.00 1.00 AOB (amoA) No. of clones 35 – 20 18 – 17 No. of OTUs (3 %) 8 – 2 1 – 1 Coverage (%) 77.10 – 90.00 94.40 – 94.10 Shannon Index 1.65 – 0.20 0.00 – 0.00 Simpson Index 0.25 – 0.90 1.00 – 1.00 Chao1 (3 %) 8.33 – 2.00 1.00 – 1.00 Anammox bacteria (16S rDNA) No. of clones 30 24 11 26 – – No. of OTUs (3 %) 7 4 5 6 – – Coverage (%) 76.70 83.30 54.50 76.90 – – Shannon Index 1.59 1.08 1.52 1.50 – – Simpson Index 0.22 0.37 0.17 0.24 – – Chao1 (3 %) 7.50 4.00 5.00 6.00 – –

(Table 3). However, no sequences related to anammox rDNA sequences were detected. The first phylotype cov- bacteria were retrieved from the Core Zone. For hzo genes ered sequences retrieved from all five positive PCR of anammox bacteria, no sequences were retrieved for any amplified samples and had 99 % identity to the cultured sample. Nitrosospira sp. Ka3 clone which was originally detected from soil in Norway (Purkhold et al. 2003). This phylotype Community characteristics of b-AOB by analyzing 16S accounted for 98 % of screened clones (94 of 96 clones) rDNA sequences and fell into the cluster 4 supported by very high boot- strapped values (98–100 %). Nitrosospira spp. in cluster 4 A total of 96 AOB-like 16S rDNA clones were sequenced were commonly found in soils and could inhabit in a wide and resulted in 2 OTUs in sample 6 and HE-Top, 1 OTU in range of pH (Stephen et al. 1998). sample 8, sample 9 and HE-Bottom (Table 4). The estimated coverage of all libraries other than HE-Top was very high (Table 4), supporting that the clone libraries constructed had Community characteristics of b-AOB by analyzing already covered the majority of AOB 16S rDNA sequences. amoA gene Compared with the results of bacterial amoA OTUs, the 16S rDNA constructed libraries had a relatively low richness but A total of 90 amoA clones sequenced and 10 OTUs were more successful amplifications achieved. The low diversity detected, with 8 OTUs from sample 6, 2 OTUs from sample of AOB detected using 16S rDNA gene was most likely due 8, and 1 OTU from sample 9 and HE-Bottom (Table 4). A to the high conserved properties of 16S rDNA gene over higher richness of the b-AOB communities was detected in bacterial amoA gene in which very high sequence similarity the Buffer and Experimental Zones. The richness of the b- among the 16S rDNA gene occur, and Nitrosospira-like 16S AOB amoA genes in sample 6 of Buffer Zone was the highest rDNA sequences were nearly identical (C99 %) to each based on several ecological calculations, namely Shannon other within this genus (Junier et al. 2010). index, Simpson index and Chao1 (Table 4). All of the four The phylogenetic tree constructed with 16S rDNA successful constructed clone libraries had high estimated sequences is shown in Fig. 2. Only two phylotypes coverage, indicating the majority of the bacterial amoA gene belonging to Nitrosospira and Nitrosomonas-like 16S sequences were sequenced.

123 Distribution patterns of ammonia-oxidizing bacteria

HE-Bottom-1 (21 clones) S8-10 (15 clones) S9-1 (24 clones) 99 Cluster 4 AJ012106. Nitrosospira sp. Ka4 AY123806. Nitrosospira sp. Ka3 100 EF019270. clone Elev 16S 452 S6-5 (19 clones) 67 Cluster 4-related 98 HE-Top-3 (5 clones) AF338208. TT140-89A

100 AY123794. Nitrosomonas sp. Nm143 Nitrosomonas sp. Nm143 lineage 76 AF338209. TT140-098-2 AF272417. Nitrosomonas communis 100 Cluster 8 AF272425. Nitrosomonas nitrosa

95 AF272418. Nitrosomonas marina AF272414. Nitrosomonas ureae 79 Cluster 6 99 S6-4 (1 clone) 100 HE-Top-2 (1 clone) AB008000. Variovorax paradoxus MBIC3839 100 AB051691. Variovorax sp. HAB-30

0.02

Fig. 2 Phylogenetic relationship of the major phylogenetic groups in 1,000 resample data sets (only values greater than 50 % are shown). the clone libraries based on AOB 16S rDNA gene using primers Branch lengths correspond to sequence differences as indicated by the NitA/NitB (*1055 bp). The numbers in the brackets are the clone scale bar. The clustering reference was defined previously (Junier number of the related phylotype. The numbers at the nodes are et al. 2008; Purkhold et al. 2000; Purkhold et al. 2003) and modified percentages that indicate the levels of bootstrap support based on in this study

Phylogenetic tree of the b-AOB amoA genes was con- Community characteristics of anammox bacteria structed with cluster classification defined by Zhang et al. by analyzing 16S rDNA (2009) and Avrahami et al. (2002). In the phylogenetic analysis (Fig. 3), the entire amoA gene sequences fell into 4 A total of 92 clone sequences through anammox bacterial clusters, which were related to Nitrosospira with 93–99 % 16S rDNA analysis were obtained from four samples and identity, but none of them was related to any Nitrososomas. 13 OTUs were identified (Table 4). Based on the Shannon From Fig. 3, 19 clones from sample 8, 18 clones from sample index, Simpson index, Chao1 and OTUs values, sample 6 9 and 17 clones from HE-Bottom were grouped into cluster 1 from surface layer sediments in Buffer Zone had the and had 98–99 % identities to the cultured Nitrosospira sp. highest diversity whereas sample 7 from low layer sedi- Ka4 which was originally retrieved from the lead contami- ments in the same zone showed the lowest diversity. In nated soil in Norway. Cluster 3a included one clone from general, estimated coverages of the four clone libraries sample 8 which was 98 % similar to an uncultured bacterium were quite high except for sample 8 (Table 4). clone from soil planted with common bean. Four individual The phylogenetic analysis of the anammox bacteria com- phylotypes were formed from sample 6 alone and were munities based on 16S rDNA sequences were constructed related to the closest amoA gene sequence representatives using the representative sequences retrieved from the Gen- Nitrosospira sp. Nsp12 (97 %) and uncultured bacterial Bank based on 3 % of DNA similarity cutoffs (Fig. 4). The clone pBru.1 (99 %). All of them were grouped into the phylogenetic tree in Fig. 4b showed the detailed Ca. Scalin- cluster 4 supported by high parsimony bootstrap values of dua cluster. It was clear that clones from the four clone 90 % (Fig. 3). Several amoA sequences from sample 6 were libraries were divided into four lineages within this cluster, 93 %–95 % identical to each other and formed a unique including Ca. Scalindua sorokinii/brodae lineage, Ca. Scal- phylotype which fell into the newly proposed cluster ME by indua arabica lineage, Ca. Scalindua wagneri lineage, and Ca. Zhang et al. (2009). Scalindua marine and freshwater related lineage.

123 Kwok-Ho Lee et al.

Fig. 3 Phylogenetic S6-16 (3 clones) relationship of the major 71 AJ298716. Nitrosospira sp. Nsp12 phylogenetic groups in the clone 83 Nitrosospira libraries based on AOB amoA AJ298687. sp. 40KI AJ298694. Nitrosospira sp. III2 gene sequences using primers Cluster 4 amoA-1F/amoA-2R (*450 bp). 51 S6-8 (4 clones) Cluster 4

S6-14 (2 clones) 16S rDNA The numbers in the brackets are 90 the clone number of the related 100 AJ388580. clone pBru.1 phylotype. The numbers at the S6-D7 (1 clone) nodes are percentages that 60 AJ298697. Nitrosospira sp. Ka4 indicate the levels of bootstrap AJ298696. Nitrosospira sp. Ka3 support based on 1,000 AY098878. clone Agb22c6 resample data sets (only values 52 Cluster 1 100 HE-Bottom-3 (17 clones) greater than 50 % are shown). S9-3-6 (18 clones) 81 Branch lengths correspond to 89 sequence differences as S8-2-5 (19 clones) indicated by the scale bar. The 66 AJ298698. Nitrosospira sp. L115 100 AJ298695. Nitrosospira sp. III7 Cluster 2 clustering reference was defined rDNA previously (Purkhold et al. AJ298723. Nitrosospira sp. O4 Cluster 2

2003; Zhang et al. 2009) and 76 S6-E2 (2 clones) 16S modified in this study 74 EU770921. clone CL147 pristine wetland 100 FJ853324. clone AOB M12 10 FJ853326. clone AOB M12 12 94 Cluster ME 99 S6-15 (17 clones) 93 EU667707. clone MAR AOB 24 83 S6-10 (6 clones) 100 EF990679. clone B2 AJ298719. Nitrosospira sp. Nsp2

99 S8-3-1 (1 clone) Cluster 3a rDNA 80 AY944215. clone PWT1-61 Cluster 3 AF272399. Nitrosomonas communis 16S Cluster 8 (out-group) AF272404. Nitrosomonas nitrosa

0.02

Discussion The predominance of the Nitrosospira over Nitroso- monas (in both amoA and 16S rDNA libraries) was Distribution and phylogeny of b-AOB detected in this study and it was consistent with the research results found in other freshwater environments in The results showed that both sample 6 and HE-Top had one which deeper sediments (5–20 cm) were dominated by clone with 98 % similarity to the isolate Nitrosomonas sp. Nitrosospira-like lineage with low potential ammonia- Nm84 retrieved from suspended particulate matter in River oxidizing activities (Coci et al. 2008; Herrmann et al. 2009; Elbe of Germany and grouped into cluster 6a based on Laanbroek and Speksnijder 2008). Junier et al. (2008) and Purkhold et al. (2003) classifica- tions. The representative isolates in cluster 6a were Nitr- Distribution and phylogeny of anammox bacteria osomonas oligotropha and Nitrosomonas ureae and both were halophilic (Purkhold et al. 2000). Both isolates were The richness of the anammox bacteria in the freshwater mostly retrieved from freshwater environments, e.g., rivers marsh was relatively high (with 4 to 7 OTUs detected in and lakes whereas some of them were recovered from soils each samples and 13 OTUs in total) compared with that with neutral pH (Ce´bron et al. 2003; Hovanec and DeLong detected in other studies like marine sponges (Mohamed 1996; Purkhold et al. 2000). The recovery of Nitroso- et al. 2009), coastal oxygen minimum zone (Gala´n et al. monas-like 16S rDNA sequences was not consistent with 2009), river estuary (Dale et al. 2009), bay sediment (Rich the results of bacterial amoA gene detected in which none et al. 2008). The relatively high richness of anammox of the Nitrosomonas-like amoA gene sequences was bacteria community detected in the Honghe freshwater observed. However, due to only one clone retrieved from marsh in this study is in agreement with the previous evi- each of the two samples, it might also be explained or even dence (Penton et al. 2006). Both studies suggested that provided further evidence that very low abundance of the anammox bacteria are widely spread and diverse in the Nitrosomonas spp. was present in this freshwater marsh. freshwater environments and might play an important role

123 Distribution patterns of ammonia-oxidizing bacteria

(a) 97 Ca. Scalindua

96 S9-H1 (3 clones) AF375995. Ca. Kuenenia stuttgartiensis DQ459989. Ca. Brocadia fulgida 72 67 AF375994. Ca. Brocadia anammoxidans

DQ301513. Ca. Jettenia asiatica Anammox bacteria 93 DQ317601. Ca. Anammoxoglobus propionicus AJ231184. Planctomyces maris (out-group)

0.05

(b) 89 EU142948 Ca. Scalindua brodae 71 AY254883 Ca. Scalindua brodae S8-B9 (2 clones) 100 AY257181 Ca. Scalindua sorokinii Scalindua

EU478631 Ca. Scalindua sp. Ca. Sorokinii/brodae 96 S7-B1 (14 clones) S8-B6 (4 clones) 99 EU478624 Ca. Scalindua arabica 99 EU478625 Ca. Scalindua arabica Scalindua arabica S6-A12 (1 clone) Ca. 70 South China Sea sediment DQ996945 53 95 S9-B10 (2 clones) S6-A5 (1 clone) S6-A8 (2 clones) 55 S8-B4 (5 clones)

51 FJ230287. clone 2G8 8 DQ647429. Xinyi River S9-H3 (13 clones) freshwater related Scalindua and Marine S7-E10 (3 clones) 60 S6-A6 (23 clones) Ca. AY254882 Ca. Scalindua wagneri S6-B2 (3 clones) 99 100 S8-B10 (1 clone) Scalindua wagneri S9-H10 (8 clones) . a

63 S7-E6 (7 clones) C

0.01

Fig. 4 Phylogenetic relationship of the major phylogenetic groups the levels of bootstrap support based on 1,000 resample data sets (a general phylogenetic tree with compressed Ca. Scalindua cluster; (only values greater than 50 % are shown). Branch lengths corre- b detailed Ca. Scalindua cluster shown) in the clone libraries based on spond to sequence differences as indicated by the scale bars. The 16S rDNA gene using primers Amx368F/Amx820R (452 bp). The clustering reference was defined previously (Dale et al. 2009; numbers in the brackets are the clone number of the related Woebken et al. 2008) with some modifications in this study phylotype. The numbers at the nodes are percentages that indicate in the ammonium transformation of freshwater ecosystems, anammox bacteria which utilize nitrite as electron acceptor even in less polluted freshwater environment like the and react with ammonium to produce dinitrogen gas (Jetten pristine and well-protected freshwater marsh in this study. et al. 1998). According to the BLAST analysis in GenBank, Samples HE-Top and HE-Bottom had the highest con- all the clones retrieved from samples HE-Top and HE- centration of ammonium and nitrite among all six samples Bottom in the Core Zone, however, were not related to any and theoretically, they could provide more substrate and identified anammox bacteria cultures in the GenBank but energy source for inhabitation of lithoautotrophic showed correlation to the uncultured bacteria or other

123 Kwok-Ho Lee et al. bacteria phyla such as Verrucomicrobia and Gracilicutes although probably at low nitrification rate and maintained a (Janssen et al. 2002). The recovery of non-Planctomyce- low level of sediment oxygen. This oxygen limited con- tales 16S rDNA sequences might suggest that the speci- dition maintained by AOB would favor the growth of ficity of the current 16S rDNA PCR primers targeting anammox bacteria which utilize the nitrite produced by anammox bacteria still needs further improvement to avoid AOB and ammonium to form dinitrogen gas and the oxy- the non-specific binding in PCR amplification reaction in gen restricted condition was indicated by 0.2 mg/L average the future (Li et al. 2010; Han et al. 2013a; 2013b). Failure dissolved oxygen measured in the sites (Schmidt et al. of PCR amplification and low detection percentage of any 2002). known anammox bacteria by different combination of The cooperation of these two groups would form a current anammox bacteria specific 16S rDNA primers stable community in the oxic-anoxic ecosystem of fresh- support the need of new and more efficient PCR primer water marsh. The coexistence of AOB and anammox design or even use the functional gene like hzo targeting bacteria were also demonstrated elsewhere (Wang et al. primers to determine the anammox bacteria diversity (Han 2013c; Wang and Gu 2013; Zhu et al. 2010). However, and Gu 2013; Humbert et al. 2009; Jiang et al. 2009;Li even low level of oxygen could inhibit or slow down the et al. 2010; Penton et al. 2006). growth of anammox bacteria as suggested by Jetten et al. However, the hzo gene as a biomarker of anammox (1998) and Penton (2009). The environmental ecophysiol- bacteria could not be amplified in any of the six samples in ogy of their existence might also account for the absence of this study according to the PCR conditions suggested by anammox bacteria 16S rDNA sequences and hzo gene Schmid et al. (2008) and thereafter any of the modified sequences in the Core Zone, which could be due to the conditions. The failure of PCR amplification using hzo presence of aquatic macrophytes in gene targeting PCR primers remained unclear and it might the zone in which the plant roots released oxygen to the be due to low abundance of the gene or absence of the hzo rhizosphere to maintain an aerobic condition (Herrmann Cluster 1 gene in anammox bacteria in the samples (Sch- et al. 2008; Laanbroek and Speksnijder 2008). mid et al. 2008). In Schmid et al. (2008) study, it was only proposed the confirmation of the hzo cluster 1 and hence Coupling of nitrification and denitrification in the Core only cluster 1 hzo was proposed to be suitable for the Zone phylogenetic analysis of the anammox bacteria communi- ties. Until recently, there are only a few published papers As shown in Table 2, a very low level of nitrate below the on PCR primers for investigation of anammox bacteria detection limit was observed in the Core Zone, suggesting communities in environmental samples using hzo gene (Li that nitrite produced by the ammonia oxidation by AOB et al. 2010; Quan et al. 2008; Schmid et al. 2008). Before was not further oxidized to nitrate, but probably lost in the the fully establishment and use of the hzo gene as the form of nitrous oxide or dinitrogen gas (Robertson et al. detection probe for anammox bacteria, 16S rDNA gene is 1995). Hence, under the aerobic conditions created by still the best for detection of this bacteria group. Recently, rhizosphere roots, nitrification–denitrification may take researchers found that hydrazine synthase gene is an place in the Core Zone of the freshwater marsh. Ruiz- effective biomarker to detect environmental anammox Rueda et al. (2009) reported a high rates of nitrification and bacteria (Harhangi et al. 2012). nitrate reduction in the rhizosphere than bulk samples. Numerous studies demonstrated that the rhizosphere of the Coexistence of anaerobic and aerobic ammonia/ plants provided a favorable niche for the colonization of ammonium oxidizing communities in freshwater marsh the nitrifying bacteria and denitrifiers (Bastviken et al. 2003; Ruiz-Rueda et al. 2009). It is still unclear about the Both anammox bacteria and b-AOB were detected in the relationship between AOB and anammox bacteria for Buffer and Experimental Zones and they might develop a partnership or competition with other nitrogen transform- partner relationship in these zones. It was firstly proposed ing microorganisms in the complex nitrogen cycle of by Schmidt et al. (2002) that both groups could be poten- freshwater marsh. tially natural partners under oxygen limited conditions with The community information of nitrogen transforming adequate supply of ammonium as a substrate for these bacteria can be potentially used as indicator for ecosystem bacteria and this proposed idea was further discussed in quality, particularly in light of anthropogenic impact. more studies later (Mohamed et al. 2009; Zhu et al. 2010). Anammox bacteria were proposed for this and it was The waterlogged sediment in the marsh created an oxic- established that dominance and presence of Ca. Scalindua anoxic interface due to the low oxygen solubility in water are a positive indication of the non- or less anthropogenic (Zhu et al. 2010). The AOB in the marsh sediment oxidize influence (Wang and Gu 2013; Li et al. 2010). Such ammonia to nitrite with the consumption of oxygen, information was also found in this study in that the 123 Distribution patterns of ammonia-oxidizing bacteria dominant anammox bacteria in the inner zone of the Chun J, Lee J-H, Jung Y, Kim M, Kim S, Kim BK, Lim Y-W (2007) reserve was Ca. Scalindua while Ca. Kuenenia was EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol detected in the outer zone of the marsh. Microbiol 57:2259–2261 Coci M, Bodelier PLE, Laanbroek HJ (2008) Epiphyton as a niche for ammonia-oxidizing bacteria: detailed comparison with benthic Conclusion and pelagic compartments in shallow freshwater lakes. Appl Environ Microbiol 74:1963–1971. doi:10.1128/aem.00694-07 Dale OR, Tobias CR, Song B (2009) Biogeographical distribution of The retrieved b-AOB 16S rRNA and amoA genes, and diverse anaerobic ammonium oxidizing (anammox) bacteria in anammox bacterial 16S rRNA gene from the freshwater Cape Fear river estuary. Environ Microbiol 11:1194–1207 marsh indicated their occurrence in the natural freshwater Dang H, Zhang X, Sun J, Li T, Zhang Z, Yang G (2008) Diversity and spatial distribution of sediment ammonia-oxidizing cre- marsh and possible role in nitrogen removal in the eco- narchaeota in response to estuarine and environmental gradients system. Niche specificity of anammox bacteria communi- in the Changjiang Estuary and East China Sea. Microbiology ties in the freshwater marsh was observed according to the 154:2084–2095. doi:10.1099/mic.0.2007/013581-0 environmental conditions and probably controlled by the Dorador C, Busekow A, Vila I, Imhoff JF, Witzel K-P (2008) Molecular analysis of enrichment cultures of ammonia oxidizers oxygen availability in different part of the marsh. However, from the Salar de Huasco, a high altitude saline wetland in anammox might be inhibited in the rhizosphere where northern Chile. Extremophiles 12:405–414 oxygen was released. Coexistence indicated that anammox Dymock D, Weightman A, Scully C, Wade W (1996) Molecular bacteria might develop a partnership with b-AOB in oxic/ analysis of microflora associated with dentoalveolar abscesses. J Clin Microbiol 34:537–542 anoxic interfaces in the marsh with sufficient ammonia for Erler DV, Eyre BD, Davison L (2008) The contribution of anammox the metabolism by the two groups. and denitrification to sediment N2 production in a surface flow constructed wetland. Environ Sci Technol 42:9144–9150 Acknowledgments This research was supported by a studentship Felsenstein J (1985) Confidence limits on phylogenies: an approach (KHL) from the Graduate School of The University of Hong Kong using the bootstrap. Evolution 39:783–791 and research grant number No. 2012B030800011 (GZ). Additional Frankland PF, Frankland GC (1890) The nitrifying process and its financial support of this project was from Environmental Toxicology specific ferment. Part I. Philosophical transactions of the Royal Education and Research Fund of this laboratory. We would like to Society of London(B) 181:107–128 thank the help and assistance of Mr. Dehui Shi from Honghe State Gala´n A, Molina V, Thamdrup B, Woebken D, Lavik G, Kuypers Farm for obtaining permission to gain access to the sampling sites, MM, Ulloa O (2009) Anammox bacteria and the anaerobic and of Miss Chunyu Zheng of Jin Zuo Yue Company for the trans- oxidation of ammonium in the oxygen minimum zone off portation and logistics provided during the sampling. We also thank northern Chile. Deep Sea Res Part II 56:1021–1031 Miss Jessie Lai and Kelly Lau for support in chemical analysis and Gopal B, Ghosh D (2008) Natural wetlands. In: Sven Erik J, Brian F Dr. Jing Wang for assistance in field sampling. (eds) Encyclopedia of Ecology, Vol 1. Academic Press, Oxford, pp 2493–2504 Conflict of interest The authors have no conflict of interest in Han P, Gu J-D (2013) More refined diversity of anammox bacteria research results report here. recovered and distribution in different ecosystems. 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