RESEARCH ARTICLE Spatial Variations of the Methanogenic Communities in the Sediments of Tropical Mangroves Hongmei Jing1, Shunyan Cheung2, Zhi Zhou3,4, Chen Wu3, Sanjay Nagarajan3, Hongbin Liu2* 1 Sanya Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, China, 2 Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China, 3 Department of Civil and Environmental Engineering, Faculty of a11111 Engineering, National University of Singapore, Singapore, Singapore, 4 Division of Environmental and Ecological Engineering and School of Civil Engineering, Purdue University, West Lafayette, Indiana, United States of America * [email protected] OPEN ACCESS Abstract Citation: Jing H, Cheung S, Zhou Z, Wu C, Methane production by methanogens in mangrove sediments is known to contribute signifi- Nagarajan S, Liu H (2016) Spatial Variations of the Methanogenic Communities in the Sediments of cantly to global warming, but studies on the shift of methanogenic community in response Tropical Mangroves. PLoS ONE 11(9): e0161065. to anthropogenic contaminations were still limited. In this study, the effect of anthropogenic doi:10.1371/journal.pone.0161065 activities in the mangrove sediments along the north and south coastlines of Singapore Editor: Yiguo Hong, CAS, CHINA were investigated by pyrosequencing of the mcrA gene. Our results showed that hydroge- Received: January 24, 2016 notrophic, acetoclastic and methylotrophic methanogens coexist in the sediments. The pre- dominance of the methylotrophic Methanosarcinales reflects the potential for high methane Accepted: July 30, 2016 production as well as the possible availability of low acetate and high methylated C-1 com- Published: September 29, 2016 pounds as substrates. A decline in the number of acetoclastic/methylotrophic methanogens Copyright: © 2016 Jing et al. This is an open in favor of hydrogenotrophic methanogens was observed along a vertical profile in Sungei access article distributed under the terms of the Changi, which was contaminated by heavy metals. The diversity of methanogens in the var- Creative Commons Attribution License, which permits unrestricted use, distribution, and ious contaminated stations was significantly different from that in a pristine St. John's reproduction in any medium, provided the original Island. The spatial variation in the methanogenic communities among the different stations author and source are credited. was more distinct than those along the vertical profiles at each station. We suggest that the Data Availability Statement: All the mcrA overall heterogeneity of the methanogenic communities residing in the tropical mangrove sequences obtained from this study were sediments might be due to the accumulated effects of temperature and concentrations of deposited in the NCBI Sequence Read Archive nitrate, cobalt, and nickel. (SRA) under the accession number of SRP068266. Funding: This work was supported by the National Natural Science Foundation of China (NSFC41406180) and a project of the Academy- Locality Science and Technology Cooperation of Sanya City, China (2014YD05) to H. Jing. H. Liu Introduction acknowledges support from the Hong Kong Methane (CH ) is a key component in the global carbon cycle. As a major green-house gas, it is Research Grants Council (RGC) via the GRF- 4 661813 and N_HKUST609/15 awards. Z. Zhou approximately 26 times more effective than CO2 in retaining heat in the atmosphere [1]. The acknowledges ®nancial support from the atmospheric CH4 inventory is currently increasing by ~0.4% per year [2]. Mangrove wetlands Singapore-Peking-Oxford Research Enterprise, and paddy fields, as well as the enteric fermentation that occurs during digestion in ruminants PLOS ONE | DOI:10.1371/journal.pone.0161065 September 29, 2016 1 / 18 Methanogens in Tropical Mangroves COY-15-EWI-RCFSA/N197-1, and a National are the most important sources of atmospheric CH4 [3,4]. Among them, the mangrove wet- University of Singapore Faculty Research lands are the largest natural source of CH4, contributing about 20% of the total annual emis- Committee grant, R-302-000-008-112. sion to the atmosphere [5,6]. Competing Interests: The authors have declared The mangrove wetlands are very productive coastal ecosystems and various anaerobic that no competing interests exist. microbial processes occur in their predominantly anoxic sediments. In these sediments, CH4 is produced during the terminal stage of anaerobic decomposition of organic matter by methano- gens [7], when the redox potential of the sediment reached to below -150 mV [8]. Methanogens are strictly anaerobic archaea and so they are very sensitive to of O2 [9]. The onset of methano- genesis primarily occurs at a shallow depth (i.e., 20–25 cm) of the sediments. The CH4 pro- duced undergoes vertical diffusive transportation from the sediment surface to the atmosphere, and horizontal transportation to the adjacent estuarine and coastal water column [10]. Natural factors such as the temperature, salinity and organic carbon content of the sediment [11] have also been shown to affect the geographical variation in the production and emission of CH4 in mangrove wetlands. In addition, several anthropogenic factors, such as disposal of sewage and agricultural runoff into the mangrove ecosystem have also been reported to enhance the emis- sion of CH4 [12]. Methanogens belong to the Euryarchaeota phylum of the Archaea domain, and consist of six phylogenetically diverse orders, Methanobacteriales, Methanococcales, Methanomicrobiales, Methanocellales, Methanopyrales and Methanosartinales, and 33 genera based on the gene sequences of 16S rRNA [13,14]. Methanogens are widely distributed in natural, strictly anaero- bic environments, such as: flooded rice fields [15]; freshwater and marine sediments [16,17]; deep-sea hydrothermal vents [18,19]; marine mud volcanoes [20]; hot springs [21]; and man- groves [22]. By far, most studies on methanogens in mangrove sediments were focused on the tropical regions. For example, Methanococcoides were important component in the Tanzanian mangrove [23] and Methanomicrobia and Methanobacteria were the two most abundant groups in the sediments of Sundarbans in India [24]; while Methanomicrobia dominated in the sediments of Guanabara Bay [25] and Sao Paulo state in Brazil [26]. In a recent study on the subtropical mangrove in Mai Po in China, groups of Methanomicrobiales, Methanosarcinales and Methanobacteriales were revealed [27]. However, knowledge about the phylogenetic com- position of methanogens has until recently been limited by the traditional culture-based proce- dures and conventional molecular techniques [28]. The recently-developed pyrosequencing technology might significantly enhance the detection capability of rare species, and when applied together with the functional mcrA gene, the complex methanogenic communities in natural anaerobic environments might be more accurately defined [29,30]. The mcrA gene, which is unique to and ubiquitous among all known methanogens [31], encodes the α-subunit of methyl coenzyme M reductase, which is the terminal enzyme involved in the methanogen- esis pathway, where methane is released [31]. The Singapore coastline harbors extensive areas of mangrove wetlands, but these ecosystems have suffered from both natural and anthropogenic disturbances in recent years following the increase in population and consequent industrialization. It is thought that the increased input of external nutrients and metals into the mangrove sediments from the adjacent areas might cause significant variations in the composition and activity of different microbial communities, especially methanogens. In order to better understand the anthropogenic and ecological impact on the methanogenic population in the tropical mangrove, sediment samples were collected from five tropical mangroves along the north and south coast of Singapore. These were Lim Chu Kang (LCK), Pulau Semakau (PS), Sungei Changi (SC), Pasir Ris Park (PRP) and St. John’s Island (SJ) (Fig 1). LCK is characterized by its strong agriculture activities; PS is the site of a new landfill; PRP is the location of the first toxic algal bloom in Singapore, which occurred in 2009, and it was shown to contain high levels of total nitrogen during our sampling PLOS ONE | DOI:10.1371/journal.pone.0161065 September 29, 2016 2 / 18 Methanogens in Tropical Mangroves Fig 1. The five mangrove sampling stations located along the Singapore coastline. Fig 1 was modified from a free picture from Wikipedia. Wikipedia has a free license "Permission is granted to copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1.2 or any later version published by the Free Software Foundation; with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts. Subject to disclaimers". doi:10.1371/journal.pone.0161065.g001 in 2012; SC is near to Changi airport and is downstream of both PRP and an old landfill site located at Sungei Punggol; and SJ, which is located far from any industrial or residential areas, was considered to be a pristine location [32]. In this study, pyrosequencing of the functional mcrA gene, which is a biomarker of methanogens, was applied to investigate the methanogenic populations residing in the tropical mangrove sediments in these various geographical condi- tions and subjected to different anthropogenic perturbations, and to elucidate the key environ- mental