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Archaea, Volume 2015, pp 1-9; doi:10.1155/2015/563414
Abstract: Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.1. IntroductionMethane is an important greenhouse gas because it is 25 times more powerful than CO2 in global warming potential (i.e., the ability of the gas to trap heat in the atmosphere) and thus plays a crucial role in climate change and carbon cycling [1, 2]. Methane emission has contributed approximately 20% to global climate change from preindustrial times [1, 3]. About 500–600 Tg of methane is emitted annually to the atmosphere of which 74% is biogenic, produced by methanogenic Archaea [4].The methanogenic Archaea (methanogens) usually occurs in highly reduced, anoxic environments such as landfills, wetlands, rice fields, rumen, and marine sediments where they serve as a terminal electron sink [5, 6]. Methanogens are strict anaerobes and the presence of oxygen leads to the formation of reactive oxygen species (ROS), which damage their cell membranes, DNA, and proteins [7, 8]. Methanogens are phylogenetically divided into 5 families within the phylum Euryarchaeota and are comprised of 31 known genera [9, 10]. Methanogens can utilize a wide range of compounds for methane production, but, in most natural systems, there are two major pathways for methanogenesis, reduction of CO2 (hydrogenotrophic methanogenesis) and cleavage of acetates (acetoclastic methanogenesis). A third pathway for methane generation is called methylotrophic methanogenesis that occurs in marine sediments and salt lakes where methane is produced from methylated compounds such as trimethylamine [11, 12].Landfill sites are the third largest source of methane. It constitutes about 30 and 24% of the anthropogenic methane production in Europe and US, respectively [4, 13]. In comparison to the western countries, the composition of municipal solid waste (MSW) in developing countries like India is higher (40–60%) in organic waste. This has more potential to emit higher GHGs (Green House Gases) per ton of MSW compared to the developed world [14]. Moreover, landfills in India are neither well planned nor engineered and are often found in low-lying open areas, where municipal waste is haphazardly and indiscriminately disposed. These sites have neither landfill lining to avoid percolation of leachate to groundwater table nor leachate collection facility. The city generates about 6000 tonnes of solid waste per day and the expected quantity of solid waste generation in Delhi would be about 12,750 tonnes per day by 2015 [15]. Due to scarcity of land in big cities, municipal authorities are using the same landfill for nearly 10–20 years. Thus, the possibility of anaerobic emission of GHGs further increases [16].Microbial decomposition, climatic conditions, MSW wastes characteristics, and landfilling operations are among the many factors that contribute to the generation of methane [2, 17]. The migration of gas and leachate away from the landfill boundaries and their release into the surrounding environment present serious environmental threats, including potential health hazards, fires and explosions, damage to vegetation, unpleasant odors, landfill settlement, ground water pollution, air pollution, and global warming [18–20].Wetlands (marshland) are the largest source of natural methane emissions contributing about 10–231 Tg methane per year accounting for 20–39% of annual global CH4 emission [4, 21]. Methanogens in the moist, anoxic (oxygen-free) wetland soil produce CH4 as they decompose dead plant material. The methane emission from wetland was increased by 7% from 2003 to 2007 [2, 19]. Methane production in wetlands is affected by the acetate supply through acetate fermentation or the CO2 reduction potential [22, 23]. The exponential increase in the rate of CH4 production with temperature is due to the availability of more substrates and is not associated with changes in the composition of methanogens [24]. Methanogens belonging to the groups Methanomicrobiales and Methanosarcinales performing acetoclastic and methylotrophic pathway were found to be dominant in landfill sites [25–27]. In acidic conditions, due to the presence of acid tolerant hydrogenotrophic methanogens, H2/CO2 is efficiently converted to methane compared to acetate, and methanogenic activity decreases with decrease in pH regardless of the substrates [28].The prokaryotic diversity in our planet dictates our planet’s ecosystems by acting as key functional drivers [29]. The understanding of the functional potential of the most individual microbial flora residing within the ecosystem is extremely limited because of our inability to isolate and culture them in laboratory conditions [30]. Since the methanogens are anaerobes and are difficult to culture, they are identified by culture independent molecular techniques like PCR amplification, denaturing gradient gel electrophoresis (DGGE), and quantitative real-time PCR, using molecular markers such as 16S rDNA genetic locus [31–34]. Hence, the present study was aimed at detecting the methanogenic Archaea inhabitants (richness) (by DGGE), identification by DNA sequencing, and quantification by qPCR in both the landfill sites of Delhi and marshland sites of Southern Assam, India.2. Material and Methods2.1. Collection of Leachate and Sediment SamplesLeachate samples were collected from three landfill sites (Bhalswa, Okhla, and Ghazipur) in the area of New Delhi, India. These sites are active landfill sites and are still in use. They do not have the leachate collection facility or landfill liner to avoid percolation of leachate to the ground water table (aquifer). Soil, sediment sample was collected from marshlands (Silcoorie Lake (Silchar), Badarpur, and Karimganj) of Southern Assam, India, in sterile falcon tubes. The details of sites along with criteria and physiochemical parameters are shown in Tables 1 and 2.Table 1: Sampling point from Delhi landfill site (Ghazipur, Bhalswa, and Okhla) and Southern Assam marshland (Silcoorie Lake (Silchar), Badarpur, and Karimganj) areas.Table 2: Chemical analysis of leachate samples obtained from three landfill and marshland sites. All parameters are in mg L−1 adapted from Ghosh et al. 2015 and Roy and Gupta 2012 [37, 38].2.2. Nucleic Acid Extraction, PCR Amplification, and CloningDNA from both landfill leachate and marshland sediment samples was extracted on the same day of sampling using Fast DNA Spin Kit for Soil (MP Biomedicals, CA, USA). DNA from the marshlands and landfill leachate was amplified using the primer set 86FWD and 1340REV (Table 3).Table 3: List of primers for PCR amplification of 16S rDNA gene and DGGE used in the present study.The amplification profile was 94°C for 5 min, 94°C for 30 s for 30 cycles, and 58°C for 1 minute, elongation at 72°C for 2 minutes, and final extension at 72°C for 10 minutes followed by a cooling step down to 4°C [35, 36]. Obtained 16S rDNA PCR products were purified by PCR purification kit (Fermentas, UK) as recommended by manufacturer protocol. PCR amplicons of 16S rDNA gene were cloned inside PTZ57R/T vector using the Insta-T/A cloning kit (Fermentas, UK) and transformed into Escherichia coli DH5α. The positive clones were selected using blue-white screening on Luria-Bertani plates containing Ampicillin (100 mg/mL), X-gal (20 mg/mL), and IPTG (100 mM). Then, positive clones were sequenced using M13 FWD primer.2.3. DNA Sequencing and Phylogenetic Analysis of 16S rDNA ClonesSequencing was performed for all the clones with the ABI prism 3130 Genetic Analyzer (Applied Biosystem Inc., CA) at Department of Biochemistry, South Campus, Delhi University. The sequences were edited to exclude the PCR primer-binding site and manually corrected with Sequence Scanner 1.0 (Applied Biosystems) and were checked further for vector contamination using the Vecscreen tool (http://www.ncbi.nlm.nih.gov/tools/vecscreen/). The sequences showing similarity with vector sequences from both ends were trimmed. Sequences were then compared with the available nucleotide database from the NCBI GenBank using the BLAST program [39]. The partial nucleotide sequences of 16S rDNA genes were submitted to NCBI under accession numbers KM041239 to KM041252 (Table 4).Table 4: List of accession numbers of the sequences submitted in NCBI and their percent similarity with database along with the sampling sites.Partial 16S rDNA sequences obtained from this study were used for similarity search in NCBI database using BLAST program. After performing BLAST, sequences showing similarity above 90% were used and aligned in MEGA software version 6.0 [40] using ClustalW. The phylogenetic relatedness among c
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