www.nature.com/scientificreports OPEN Comparison of community structures of Candidatus Methylomirabilis oxyfera-like Received: 09 March 2016 Accepted: 20 April 2016 bacteria of NC10 phylum in Published: 09 May 2016 different freshwater habitats Li-dong Shen1,2, Hong-sheng Wu1,2, Zhi-qiu Gao3,4, Xu Liu2 & Ji Li2 Methane oxidation coupled to nitrite reduction is mediated by ‘Candidatus Methylomirabilis oxyfera’ (M. oxyfera), which belongs to the NC10 phylum. In this study, the community composition and diversity of M. oxyfera-like bacteria of NC10 phylum were examined and compared in four different freshwater habitats, including reservoir sediments (RS), pond sediments (PS), wetland sediments (WS) and paddy soils (PAS), by using Illumina-based 16S rRNA gene sequencing. The recovered NC10-related sequences accounted for 0.4–2.5% of the 16S rRNA pool in the examined habitats, and the highest percentage was found in WS. The diversity of NC10 bacteria were the highest in RS, medium in WS, and lowest in PS and PAS. The observed number of OTUs (operational taxonomic unit; at 3% cut-off) were 97, 46, 61 and 40, respectively, in RS, PS, WS and PAS. A heterogeneous distribution of NC10 bacterial communities was observed in the examined habitats, though group B members were the dominant bacteria in each habitat. The copy numbers of NC10 bacterial 16S rRNA genes ranged between 5.8 × 106 and 3.2 × 107 copies g−1 sediment/soil in the examined habitats. These results are helpful for a systematic understanding of NC10 bacterial communities in different types of freshwater habitats. Methane (CH4) is the most important greenhouse gas after carbon dioxide, and responsible for ~20% of the current greenhouse effect1. Freshwater habitats like natural wetlands are an important source of atmospheric methane1,2. In the freshwater habitats, aerobic methane oxidation using oxygen as electron acceptor is the most important biological sink of methane, but the role of alternative electron donors is not well known3. Recently, evidence showed that methane oxidation coupled to nitrite reduction may also play an important role in reducing methane emissions from different freshwater habitats4–10. Methane oxidation coupled to nitrite reduction was first demonstrated in an enrichment culture from freshwater sediments11–13. The organism responsible for this reaction has been identified as Candidatus Methylomirabilis oxyfera (M. oxyfera), which belongs to the NC10 phylum14. This phylum was proposed by Rappe and Giovannoni15 based on 16S rRNA gene sequences recovered from flooded caves, and has no members in pure culture. Based on 16S rRNA gene or functional gene clone libraries using specific primers, the commu- nity composition and diversity of NC10 bacteria in different freshwater habitats have previously been described separately, primarily including lakes4,6,16, rivers17, wetlands7,9,10,18 and paddy fields8,19,20. Given the potential role of NC10 bacteria in reducing methane emissions21,22, it is therefore imperative to examine and compare their 1Collaborative Innovation Center on Forecast and Evaluation of Meteorological Disasters, Jiangsu Key Laboratory of Agricultural Meteorology, College of Applied Meteorology, Nanjing University of Information Science and Technology, Nanjing, 210044, China. 2Department of Agricultural Resource and Environment, College of Applied Meteorology, Nanjing University of Information Science and Technology, Nanjing 210044, China. 3State Key Laboratory of Atmospheric Boundary Layer Physics and Atmospheric Chemistry, Institute of Atmospheric Physics, Chinese Academy of Science, Beijing, China. 4College of Geophysics and Remote Sensing, Nanjing University of Information Science and Technology, Nanjing 210044, China. Correspondence and requests for materials should be addressed to L.-D.S. (email: [email protected]) SCIENTIFIC REPORTS | 6:25647 | DOI: 10.1038/srep25647 1 www.nature.com/scientificreports/ community composition and diversity in different freshwater habitats to determine whether these habitats can influence their community structures. The Illumina MiSeq platform provides researchers with a scalable, high-throughput and streamlined sequenc- ing platform to survey microbial communities from environmental samples23. This technique has been widely used to study microbial communities in various freshwater habitats, and have changed our understanding of microbial diversity in the environment24–26. The Illumina-based 16S rRNA gene sequencing has high depth cov- erage of microbial diversity and can detect both abundant and rare microbes25,27,28, and thus is a more powerful method than the clone libraries for evaluation of microbial communities. However, until now, only Lu et al.29 and Shen et al.30 have applied Illumina-based 16S rRNA gene sequencing technology to examine the community structures of NC10 bacteria, and the latter showed an unexpected high diversity of NC10 bacteria at species level in agricultural soils. To the best of our knowledge, no study has used this technique to compare the community composition and diversity of NC10 bacteria between different natural habitats. Knowledge from comparisons of NC10 bacterial community structures in different freshwater habitats may identify habitat-specific adaptations in their physiology and evolution. It can also reveal whether certain NC10 bacterial taxa tend to be detected only in one habitat or co-occur in different habitats, which has implications for their biogeography and ecology. In the present study, an improved dual-indexing amplification and sequencing approach developed by Fadrosh et al.23 was used to assess the community composition and diversity of NC10 bacterial communities in different freshwater habitats. Four different freshwater habitats were selected in this study, including the reservoir sediments, pond sediments, wetland sediments and paddy soils. We hypothesized that these habitats harbored unique NC10 bacterial assemblages. Materials and Methods Site description and sample collection. Different freshwater sediments/soils were selected in the cur- rent study, including the reservoir sediments (RS), pond sediments (PS), wetland sediments (WS) and paddy soils (PAS). These sediments were permanently or semi-permanently submerged by water, which can create anoxic conditions for the NC10 bacteria. Three RS samples (RS1, RS2 and RS3) were collected from the Jiulonghu Reservoir, which is located in Zhejiang Province, China. The upper 15 cm sediments were collected from this reservoir using box-cores in July 2015. Four PS samples (PS1, PS2, PS3 and PS4) were collected from four aqua- culture ponds that are located in Jiangsu Province, China. The upper 15 cm sediments were collected from these ponds using box-cores in November 2014 as previously described31. Four sediment samples (WS1, WS2, WS3 and WS4) were collected from a natural freshwater wetland, the Baguazhou Wetland, located in Jiangsu Province, China. The upper 15 sediments were collected from this wetland system using a stainless steel ring sampler in January 2015 as previously described32. Three soil samples were collected from a paddy field that subject to long-term fertilization in Jiangsu Province. The soil layers of 0–10 cm (PAS10), 20–30 cm (PAS30) and 40–50 cm (PAS50) were collected from the paddy field using a stainless steel ring sampler in June 2015. All the collected sediment/soil samples were transferred immediately to sterile plastic bags, and transported to the laboratory on ice within 12 h. The collected samples were subsequently divided into two parts. The first part was stored at 4°C for physicochemical analysis, and the other part was stored at − 20 °C for later molecular analysis. Chemical analysis. The sediment/soil pH was determined after mixing the sediment/soil with deionized water at a ratio (sediment/soil: water) of 1:2.5. Sediment/soil ammonium and nitrate were extracted from the sediment/soil using 2 M KCl as previously described33, and the extracted ammonium and nitrate were determined 34,35 according to the reported studies . The sediment/soil organic carbon content was determined by the K2Cr2O7 oxidation method36. DNA isolation. Sediment/soil DNA was extracted using a Power Soil DNA kit (Mo Bio Laboratories, Carlsbad, California, USA) according to the manufacturer’s instructions. Approximately 0.25 g homogenized sediment/soil was used for DNA isolation. The quality of the extracted DNA was evaluated on 1% agarose gel, and the concentration of DNA was measured with a NanoDrop spectrophotometer (ND-1000; Isogen Life Science, the Netherlands). Illumina-based 16S rRNA gene sequencing. High-throughput sequencing of bacterial 16S rRNA genes was performed on the Illumina MiSeq platform (Hangzhou Guhe Information and Technology Co., Ltd., Zhejiang, China). Briefly, the V3-V4 region of bacterial 16S rRNA genes was amplified by the primer 319f (5′ -ACTCCTACGGGAGGCAGCAG-3′ ) and primer 806r (5′ -GGACTACHVGGGTWTCTAAT-3′ ) as previ- ously described23,30. Briefly, PCRs were performed in a 30 μ l mixture containing 0.5 μ l Dimethyl sulfoxide, 1.0 μ l forward primer (10 mM), 1.0 μ l reverse primer (10 mM), 5.0 μ l DNA template, and 15.0 μ l Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB). The thermal program was as follows23,30: 98 °C for 30 s; 30 cycles of 98 °C for 15 s, 58 °C for 15 s, and 72 °C for 15 s; and a final extension at 72 °C for 1 min. PCR products were purified using an agarose gel DNA purification kit (Qiagen, Chatsworth, California, USA) and then were subjected to Illumina MiSeq sequencing (2 × 300 bp). Analysis of Mi-Seq sequencing data was conducted using Quantitative Insights Into Microbial Ecology (QIIME, www.qiime.org) according to Shen et al.30,31. High-quality NC10 phy- lum bacterial sequences were further confirmed by aligning these sequences with the reported NC10 sequences deposited in GenBank using the BLAST search engine30. Representative sequences for each operational taxo- nomic unit (OTU) as defined by 97% sequence identity were obtained for further analysis. The coverage of NC10 bacteria in each sample was calculated as C = [1 − (n1/N)] × 100, where n1 is the number of unique OTUs and N is the total number of sequences in the sample.