See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/321071685

Molecular detection of Methylotrophs from an

Indian landfill site and their potential for
biofuel production

Article in GlobalNEST International Journal · November 2017

CITATIONS READS

0 10

2 authors, including:

Subhrangsu Maitra
Jawaharlal Nehru University
27 PUBLICATIONS 21 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Want to collaborate with sombody with a spectrophotometer with a flow through cuvette for online
detection of instability in recombinant plasmid based fermentations View project

UPOE -II -5 View project

All content following this page was uploaded by Subhrangsu Maitra on 16 November 2017.

The user has requested enhancement of the downloaded file.

 Global NEST Journal, Vol 19, No 3, pp 533-539
Copyright© 2017 Global NEST
Printed in Greece. All rights reserved

Molecular detection of Methylotrophs from an Indian landfill site

and their potential for biofuel production
Yadava S.1,* and Maitra S.S.2
1Department of Biotechnology, Assam University, Silchar, PIN-788011

2School of Biotechnology, Jawaharlal Nehru University, New Delhi, PIN-110067

Received: 09/06/2017, Accepted: 19/09/2017, Available online: 24/10/2017

*to whom all correspondence should be addressed: e-mail: [email protected]

Abstract and generation of greenhouse gases like methane (Yadav

Emission of CH4 from landfills is a major cause of concern et al., 2015). Methane is an important greenhouse gas,
as CH4 is twenty four times more potent than CO 2, as a because it is 25 times more potent than CO2 in global
greenhouse gas. However, landfills also harbor a group of warming (Yadav et al., 2014). Despite directives from the
bacteria called methanotrophs, which can oxidize CH4. Honorable Supreme Court and the MSW Rules 2000 of
They can be used for in situ bioremediation to reduce Government of India, Municipal Corporation of Delhi has so
methane emissions. They can also be used for production far failed to address the issue of Landfill Gas(LFG) recovery
of methanol or renewable diesel, utilizing methane in (mixture of CO2 ,CH4 gas): which is causing great harm to
natural gas or biogas. Methanotrophs are a subgroup of environment and leading to loss of a potential source of
methylotrophs. We used molecular techniques for energy (Talyan et al., 2008; Talyan et al., 2007).
detection of methylotrophs in samples from a landfill in Access to clean, affordable and reliable energy has been a
New Delhi. We could detect five methylotrophs. Isolation major requirement of the world’s increasing population
and efficiency in methanotrophy of these bacteria is and economic growth. Our use of energy in the twenty-first
undergoing now. century must be sustainable. Solar and water-based energy
Keywords: Methylotrophs, Molecular detection, Municipal generation and engineering of microbes to produce
Solid Waste leachate biofuels, are a few examples of alternatives (Chu and
Majumdar, 2012).
1. Introduction
Diminishing oil reserves and climate-changing greenhouse
Climate change is one of the most important challenges gas emissions have led to calls for clean and renewable
that the humankind is facing in this era. It is related to liquid fuels (Hu et al. 2016). Currently, India contributes
forest and land degradation, freshwater shortage, food about 5% of total global CO2 emissions. To prevent CO2
security and air-water pollution. According to emissions to peak quickly, part of India’s new energy needs
Intergovernmental Panel on Climate Change (IPCC) report, must come from low-carbon technologies. However, India
the global mean temperature may increase between 1.4 to had only 60 GW of low-carbon capacity installed by the end
5.8 °C by 2100 (Mitigation, 2011). The impact of this rise in of 2014, and only 3 GW of solar power. A more robust
temperature would be particularly severe, in the tropical electrical grid and a dramatic rise in renewable energy
areas, which mainly consist of developing countries sources are required to achieve the target (Jackson et al.,
including India. The climate change issue for developing 2015).
countries is correlated with pace of sustainable
development (Sathaye et al. 2006). At present India’s Methane (CH4) is the second most important atmospheric
requires plans for sustainable development and she must greenhouse gas after CO2 (Cai et al. 2016) and is believed to
address many climate change concerns. These conscious account for 17% of global warming (Collins et al. 2013).
policy decisions may prove helpful for obtaining climate- Although most sources and sinks of methane have been
friendly sustainable development model (Sharma et al. identified, their relative contributions to atmospheric
2006; Kumar et al. 2009). methane levels are uncertain (Houghton et al., 2001; Yvon-
Durocher et al., 2014; Bridgham et al., 2013; Kirschke et al.,
Municipal Solid Waste (MSW) management has remained 2013). About 500–600 Tg of methane is emitted annually
one of the most neglected areas of the municipal service to the atmosphere of which 74% is biogenic (Solomon
delivery in Delhi, India. About 70–80% of generated MSW 2007; Jackson et al., 2015).
is collected, 9% of which is treated through composting and
rest is disposed in uncontrolled open landfills. In absence Landfill sites are among the largest anthropogenic source
of leachate and landfill gas collection systems, these of methane. It constitutes 30 and 24% of the anthropogenic
landfills are a major source of groundwater contamination CH4 production in Europe and the US, respectively

Yadava S. and Maitra S.S. (2017), Molecular detection of Methylotrophs from an Indian landfill site and their potential for biofuel
production, Global NEST Journal, 19(3), 533-539.
534 YADAVA and MAITRA

(Solomon, 2007; Mitigation, 2011). In comparison to the 28°37’22.4”N-77°19”25.7”E and the pH of the sample was
western countries, the composition of organic waste in in between 7.4-8.4.
municipal solid waste (MSW) of developing countries like
2.2. Nucleic acid extraction, PCR amplification and
India has been higher (40% - 60%). This gives it more
cloning
potential to emit higher GHGs from per ton of MSW
compared to the developed world (Mayumi et al., 2013). DNA from both landfill leachate samples was extracted on
Moreover, landfills in India are neither planned nor the same day of sampling, using Fast DNA Spin Kit for soil
engineered. They are often found in low-lying open areas, (MP Biomedicals, CA, USA). DNA from leachate was
where municipal waste is haphazardly and indiscriminately amplified using the universal primer set 27FWD and
disposed. These sites neither have landfill lining to avoid 1492REV. The amplification profile was 94 °C for 5min, 94
percolation of leachate to groundwater table nor leachate °C for 30s for 30 cycles, 55 °C for 1minute, elongation at 72
collection facility (Yadav et al., 2015). The city of New Delhi °C for 2 minute and final extension at 72 °C for 10 minutes
generates about 9000 tons of solid waste per day (Gupta et followed by a cooling step down to 4 °C. 16S rDNA gene
al., 2007). Due to scarcity of land in big cities, municipal specific PCR products, thus obtained, were purified by PCR
authorities are using same landfill for nearly 10 - 20 years. purification kit (Fermentas, UK) as recommended by
Thus, the possibility of anaerobic digestion of MSW and manufacturer’s protocol. PCR amplicons of 16S rDNA gene
emission of greenhouse gas (GHGs) further increase (Rawat were cloned inside PTZ57R/T vector using the Insta-T/A
and Ramanathan, 2011). cloning kit (Fermentas, UK) and transformed into
Microbial decomposition, climatic conditions, refuse Escherichia coli DH5𝛼. The positive clones were selected
characteristics and land-filling operations are among the using blue-white screening on Luria-Bertani plates
many factors that contribute to the generation of methane containing Ampicillin (100mg/ml), X-gal (20mg/ml), and
(Talyan et al., 2007; Bridgham et al., 2013). The migration IPTG (100 mM). Then positive clones were sequenced using
of gas and leachate away from the landfill boundaries and M13 FWD primer.
their release into the surrounding environment presents 2.3. DNA Sequencing and Phylogenetic Analysis of 16S
serious environmental concerns (Hansen et al., 1998; rDNA clones
Bloom et al., 2010; Singh and Mittal, 2011). Leachate
harbors huge prokaryotic diversity, which yet remains to be Sequencing was performed for all the clones with the ABI
discovered. Characterizing microbial diversity of that prism 3130 Genetic Analyzer (Applied Biosystem Inc., CA)
mediates carbon and methane flux in landfills, is critical for at the Department of Biochemistry, South Campus, Delhi
predicting their role and responses to climate changes University. The sequences were edited to exclude the PCR
(Bardgett and van der Putten, 2014). primer-binding site and manually corrected with Sequence
Scanner 1.0 (Applied Biosystems) and were checked further
Methanotrophs use methane as their sole carbon source for vector contamination using the Vecscreen tool
and directly convert methane into cellular compounds or (http://www.ncbi.nlm.nih.gov/tools/vecscreen/).
transform it into a substrate that drives processes via
interaction with other microbes. The biological conversion The sequences showing similarity with vector sequences
of methane has become a topic of intense interest. In 2013 from both ends were trimmed. Sequences were then
the Advanced Research Projects Agency (ARPA-E) ,within compared with the available nucleotide database from the
the U.S. Department of Energy, granted US $34 million NCBI GenBank using the BLAST program (Altschul et al.,
worth of funding directed toward research for converting 1997). The partial nucleotide sequences of 16S rDNA genes
methane into liquid fuels (Strong et al., 2015). Bacteria like were submitted to NCBI under accession numbers
Methylosinus trichosporium, have the capacity to oxidize KM041243 to KM041247 (Table 1). Partial 16S rDNA
methane. However, processes for microbial conversion of sequences obtained from this study were used for
methane to biodiesel are marred by low productivity, slow similarity search in NCBI database using BLAST program.
growth rate and undesirable substances in the lipid After performing BLAST, sequences showing similarity
accumulated by the bacteria. Therefore, there is need for above 90% were used and aligned in MEGA software
bio-prospecting for a better methanotroph. version 6.0 (Tamura et al., 2013) using ClustalW. The
phylogenetic relatedness among clones was estimated
In the present study, a PCR based study was performed for using the Maximum Likelihood Tree using Kimura K2P+G
detection and community fingerprinting of methylotrophs model with 2000 bootstrap value (Kimura, 1980). For
present in the MSW leachate from a landfill site of Delhi, model selection Bayesian analysis was performed and the
India, targeting 16S rDNA sequences with methylotroph model with lowest BIC value (i.e., i.e. 9109.105) was chosen
specific primers. for tree construction. All positions containing gaps and
2. Materials and Methods missing data were eliminated from the dataset (complete
deletion option).The phylogenetic analysis was carried out
2.1. Sampling using MEGA software version 6.0 (Tamura et al., 2013)
Samples were collected from the Ghazipur landfill site of (Figure 1).
Delhi. It is situated in east Delhi and is the oldest and largest 2.4. Denaturing Gradient Gel Electrophoresis
landfill of Delhi. It was established in the year 1984 and
spans 29.6 hectares in area. It has received about 2200 TPD For denaturing gradient gel electrophoresis, genomic DNA
waste till now. The GPS location of sampling point was extracted from the landfill leachate was amplified using
MOLECULAR DETECTION OF METHYLOTROPHS FROM AN INDIAN LANDFILL SITE 535

primer 27FWD and GC 968 which gave a product length of denaturant contained 7 M urea and 40% Formamide.
about 700 bp. DGGE was performed with a D-Code Electrophoresis was initially run at 200 V for 10 min at 60°C,
universal mutation detection system (Biorad, Hercules, CA, and afterwards at 130 V for 4 hours. After electrophoresis,
USA), using 16 cm by 16 cm and one mm gels. PCR products the gel was silver stained and scanned under white light
were loaded onto 7% (w/v) polyacrylamide gels. The using Gel doc (Biorad). DGGE gel was further analyzed using
polyacrylamide gels (Bis-Acrylamide, 37.5:1) were made Gel2K software (Svein Norland, Department of Biology-
with denaturing gradients ranging from 40 to 60%. 100% University of Bergen, Norway) (Figure 2).
Table 1. Methylotrophic 16S rDNA amplicon sequences with accession numbers submitted to NCBI.
Sequence ID Size Accession no. Location Organism name Closest relative
METG2_16S rDNA 701 Nt KM041243 Ghazipur landfill Methylobacillus Methylobacillus
flagellatus flagellatus
METG3_16S rDNA 705 Nt KM041244 Ghazipur landfill Methylotenera Methylobacillus
mobilis flagellatus
METG4_16S rDNA 719 Nt KM041245 Ghazipur landfill Methylobacillus Methylobacillus
gramineus flagellatus
METG6_16S rDNA 729 Nt KM041246 Ghazipur landfill Methylobacillus Methylobacillus
arboreus flagellatus
METG7_16S rDNA 637 Nt KM041247 Ghazipur landfill Methylovorus Methylobacillus
glucosetrophus flagellatus

Figure 1. The phylogenetic relationship of 25 partial 16S rDNA sequences (the confirmed 5 sequences of clones
generated in this study, recovered from Ghazipur landfill site Delhi ,marked with black triangle) was inferred by the ML
method using K2P+G parameter model with 2000 bootstrap replicates using the MEGA 6 tree building program

Figure 2. Showing Jaccard cluster analysis of methylotrophic community based on 16S rDNA amplicons from samples
obtained from five locations of Ghazipur landfill site Delhi
536 YADAVA and MAITRA

3. Results and Discussion excess reducing equivalents through the respiratory chain.
Further in 2015, Gilman et al., found that this strain has
3.1. Identification of methanotrophs present in landfill site
both high growth rate and carbon conversion efficiency
Delhi using 16S rDNA sequencing
and emphasized on its use for commercial methane
Present study investigates presence of methylotrophs in bioconversion to value-added products such as fuels and
landfill leachate of Ghazipur landfill site. To the best of our chemicals (Gilman et al., 2015).
knowledge this is first study where molecular analysis of
3.2. Diversity of Methylotropic Bacteria by Denaturing
methylotrophs/methanotrophs have been performed and
Gradient Gel Electrophoresis
their presence reported from a Indian landfill site.
Phylogenetic analysis of 16S rDNA clones indicate the Abundance and diversity of methylotrophic bacteria were
presence of methanotrophs belonging to the domain studied using denaturing gradient gel electrophoresis in
bacteria, phylum proteobacteria, order Methanophilales, leachate samples collected at five different locations at
genus methanobacillus in the Ghazipur landfill site of Delhi. Ghazipur landfill site, Delhi. 16S rDNA gene amplicons used
Sequence METG2_16S_rDNA matched with for cloning were analyzed on the DGGE gel for estimation
Methylobacillus flagellatus strain belonging to order of their richness in respective samples. Banding patterns of
Methanophilales. METG3_16S_rDNA matched with 16S rDNA amplicons obtained from all five leachate
Methylotenera mobilis. MET4_16S_rDNA matched with samples of the Ghazipur landfill site were compared using
Methylobacillus gramineus and METG6_16_rDNA matched Gel 2K software and the analysis of DGGE image revealed
with Methylobacillus arboreus, which is a obligate the presence of total 23 bands Figure 2.
methanol-utilizing Gram-negative, asporogenous, motile There are some unique bands in each lane, which indicates
rod that occur singly, in pairs or in small groups. They the variation of methylotrophic community residing in
oxidize methanol by PQQ-MDH to formaldehyde and those particular samples. Cluster analysis of bands using
assimilate it via the RuMP pathway (Gogleva et al., 2011). Jaccard analysis indicated the presence of two main
Sequence METG7_16S_rDNA matched with Methylovorus clusters on the basis of number of similarity and DGGE
glucosetrophus. All them showed 99% to 96% similarity to bands in Figure 2. In first cluster samples GZ3, GZ4 and GZ5
Methylobacillus flagellatus. The family Methylophilaceae sample clustered together showing similar community
includes all four described genera (Methylophilus, structure and diversity. In second cluster samples GZ1 and
Methylovorus, Methylobacillus and Methylotenera). All GZ2 of leachate clustered together. It indicates that
represent obligate or restrictive facultative methylotropy. methylotrophic community structure inhabiting in all five
Methylobacillus flagellatus belongs to the leachate samples were similar. In terms of richness
Betaproteobacteria class and is most closely related. regarding number of bands, GZ4 samples has maximum 13
Methylobacillus flagellatus strain KT utilizes methanol and bands followed by GZ2, GZ1, GZ2 and GZ5 having 12,11 and
methylated amines as the sole sources of carbon and 10 bands respectively. Methylotrophic community
energy and is classified as an obligate methylotroph. The diversity present in all five samples on the basis of band
strain was isolated from a metropolitan sewer system and pattern were found to be uniform in the Ghazipur landfill
selected as a prospective industrial strain due to its high site.
growth rates on methanol, high tolerance to methanol and
3.3. Culture independent molecular analysis of
formaldehyde, high biomass yield, and high coefficient of
Methylotrophs
conversion of methanol into biomass (Baev et al., 1992;
Chistoserdova et al., 2007). Methylotrophy is the metabolic Methylotrophs/methanotrops have gained attention since
capacity to grow on reduced carbon compounds such as last decades, as they have considerable potential for the
methane, methanol, methylated amines, etc. oxidation of methane (Schimel, 2000; Singh, 2011). The
Methylotrophy functions in Methylobacillus flagellatus, slow growth rate of methanotrophs hinders their study by
(Chistoserdova et al., 2007). This can also be a type I conventional culture dependent techniques. Methods such
methanotroph belonging to gamma-proteobacteria. as the MPN technique, FISH can be biased by selective
Benstead et al. 1998, found that methanol promotes culture conditions or require too much manual effort.
atmospheric methane oxidation by methanotrophic Therefore, PCR based molecular techniques are widely
cultures and in soil. Methanol enhances activity of used for their identification and diversity analysis.
methanotrophic activity in soils by acting either as Methanotrophs are extensively studied in a wide variety of
substrate for growth and source of energy production or as environments like rice paddy fields, landfill cover soil etc.
reducing agent that are required for the continued because of their critical role in the global carbon cycle and
oxidation of atmospheric methane(Benstead et al., 1998; methane mitigation (Cai et al., 2016; Han et al., 2009;
Hanson and Hanson, 1996). Eshinimaev et al. 2002 found Singh, 2011; Su et al., 2014).
that alkaliphilic methanotroph Methylomicrobium Culture-independent surveys showed that methanotrophs
buryatense 5B is capable of growing at high methanol belonging to family Methylophilaceae are ubiquitous,
concentrations (up to 1.75 M). They hypothesized that the thriving in a variety of natural as well as man-made
high methanol tolerance of M. buryatense 5B is due to the environments. (Murrell et al., 1998; Chen et al., 2007; Han
utilization of formaldehyde for the synthesis of sucrose, et al., 2009; Musenze et al., 2016; Lee et al., 2015; Su et al.,
glycogen, and the glycoprotein and to the oxidation of 2014). In present study we used 16S rDNA based cloning-
MOLECULAR DETECTION OF METHYLOTROPHS FROM AN INDIAN LANDFILL SITE 537

sequencing and DGGE for identification and diversity Methanotrophs are a special group of bacteria within
analysis of methylotrophs/methanotrophs present in the methylotrophs. Methanotrophs can survive on methane as
leachate samples of Ghazipur, Delhi. Novel Type 1 obligate sole carbon source for both growth and energy generation.
methanotrophs belonging to class gamma proteobacteria We have identified a few methylotrophs. Research is
has been also found to be associated with leachate samples continuing for their isolation. Study is being done whether
of Ghazipur landfill site which can utilize methane and they could survive on methane alone. If that is case, they
formaldehyde as energy source (Chistoserdova et al., 2007; qualify for being methanotrophs as well.
Gogleva et al., 2011). Acknowledgment
3.4. Methylotrophic potential for methane mitigation and Dr. S. S. Maitra is grateful to the Department of Science and
biofuel production Technology (DST), Government of India( under PURSE program)
Technology for conversion of natural gas/landfill gas to and Jawaharlal Nehru University (under UOPE-II ,UGC) , for
providing grants.
liquid fuel is highly desirable. Current chemical conversion
of natural gas to liquid fuels is not economically References
competitive. Bioconversion is a promising alternative Altschul S.F.l., Madden T.L., Schaffer A.A., Zhang J., Zhang Z.,
because of high specificity and high process energy Miller W. and Lipman D.J. (1997), Gapped BLAST and PSI-
efficiency under very mild conditions. Biotechnology aims BLAST: a new generation of protein database search
to provide sustainable solutions for the provision of fuels, programs, Nucleic Acids Res, 25(17), 3389-3402.
chemicals and materials (Nielsen et al., 2014). Global Baev M., Schklyar N., Chistoserdova L., Chistoserdov A., Polanuer
climate change linked to the accumulation of greenhouse B., Tsygankov Y. and Sterkin V. (1992), Growth of the obligate
gases has caused concerns regarding the use of fossil fuels methylotroph Methylobacillus flagellatum under stationary
as the major energy source. To mitigate climate change and nonstationary conditions during continuous cultivation,
while keeping energy supply sustainable, one solution is to Biotechnology and Bioengineering, 39(6), 688-695.
rely on the ability of microorganisms to use renewable Bardgett R.D. and van der Putten W.H. (2014), Belowground
resources for biofuel synthesis (Liao et al., 2016; Singh, biodiversity and ecosystem functioning, Nature, 515(7528),
2011). To reduce or avoid collateral emissions, raw 505-511. doi:10.1038/nature13855
materials for sustainable biofuel production should come Benstead J., King G. and Williams H. (1998), Methanol promotes
from atmospheric CO2 or from carbon that is otherwise atmospheric methane oxidation by methanotrophic cultures
released as GHGs, without being efficiently utilized (Strong and soils, Applied and Environmental Microbiology, 64(3),
et al., 2015). 1091-1098.
As methane is a more potent GHG than CO2 (Yvon-Durocher Bloom A.A., Palmer P.I., Fraser A., Reay D.S. and Frankenberg C.
(2010), Large-scale controls of methanogenesis inferred from
et al., 2014), there is increasing pressure to find ways of
methane and gravity spaceborne data, Science, 327(5963),
more efficient use of methane as a carbon source.
322-325.
Methane produced from landfill or by anaerobic digestion
Bridgham S.D., Cadillo‐Quiroz H., Keller J.K. and Zhuang Q. (2013),
of various organic wastes or from “stranded natural gas”
Methane emissions from wetlands: biogeochemical,
can be exploited as feeds for production of alternate bio-
microbial, and modeling perspectives from local to global
fuel (Conrado and Gonzalez, 2014; Peralta-Yahya et al.,
scales, Global change biology, 19(5), 1325-1346.
2012). New technologies for effective conversion of
Cai Y., Zheng Y., Bodelier P.L., Conrad R. and Jia Z. (2016)
flared/waste sources of methane into chemical
Conventional methanotrophs are responsible for atmospheric
compounds, including next-generation fuels, have become
methane oxidation in paddy soils, Nature communications, 7.
active matter of research. (Kalyuzhnaya et al., 2013). In
doi:10.1038/ncomms11728
literature, there is report that Methylotrophus capsulatus,
Chen Y., Dumont M.G., Cébron A. and Murrell J.C. (2007),
Methylosinus trichosporium can convert methane and
Identification of active methanotrophs in a landfill cover soil
oxygen to methanol (Hwang et al., 2014). through detection of expression of 16S rRNA and functional
Microbial methane oxidation acts as the only known genes, Environmental Microbiology, 9(11), 2855-2869.
biological sink of methane, as methanotrophs limit the Chistoserdova L., Lapidus A., Han C., Goodwin L., Saunders L.,
release of methane from many methanogenic areas. Brettin T., Tapia R., Gilna P., Lucas S. and Richardson P.M.
Methanotrophs represent a potential biological platform (2007), Genome of Methylobacillus flagellatus, molecular
for methane-based biocatalysis. basis for obligate methylotrophy, and polyphyletic origin of
16S rDNA sequencing revealed presence of Methylobacillus methylotrophy, Journal of Bacteriology, 189(11), 4020-4027.
Chu S. and Majumdar A. (2012), Opportunities and challenges for
flagellatus strain KT in Ghazipur landfill site, Delhi. It utilizes
a sustainable energy future, Nature, 488(7411), 294-303.
methanol and methylated amines as the sole source of
Collins M., Knutti R., Arblaster J., Dufresne J., Fichefet T.,
carbon and energy. Because of its higher growth rate,
Friedlingstein P., Gao X., Gutowski W., Johns T. and Krinner G.
tolerance to methanol and high coefficient of conversion of
(2013), Contribution of working group I to the fifth
methanol into biomass can be utilized in scale up process
assessment report of the intergovernmental panel on climate
for production of methanol (alternate biofuel) using MSW
change. Climate Change
leachate as renewable energy source.
Conrado R.J. and Gonzalez R. (2014), Envisioning the
4. Conclusion bioconversion of Methane to liquid fuels, Science, 343(6171),
621-623.
538 YADAVA and MAITRA

doi:10.1038/nature13164 Kimura M. (1980), A simple method for estimating evolutionary

http://www.nature.com/nature/journal/v507/n7493/abs/na rates of base substitutions through comparative studies of
ture13164.html#supplementary-information nucleotide sequences, Journal of Molecular Evolution, 16(2),
doi:10.1038/ngeo1955.(http://www.nature.com/ngeo/journ 111-120.
al/v6/n10/abs/ngeo1955.html#supplementary-information). Kirschke S., Bousquet P., Ciais P., Saunois M., Canadell J.G.,
doi:10.1038/nrmicro.2016.32 Dlugokencky E.J., Bergamaschi P., Bergmann D., Blake D.R.,
Edenhofer, Ottmar, et al. "IPCC special report on renewable Bruhwiler L., Cameron-Smith P., Castaldi S., Chevallier F., Feng
energy sources and climate change mitigation." Prepared By L., Fraser A., Heimann M., Hodson E.L., Houweling S., Josse B.,
Working Group III of the Intergovernmental Panel on Climate Fraser P.J., Krummel P.B., Lamarque J-F, Langenfelds R.L., Le
Change, Cambridge University Press, Cambridge, UK (2011). Quere C., Naik V., O'Doherty S., Palmer P.I., Pison I., Plummer
Eshinimaev B.T., Khmelenina V.N., Sakharovskii V.G., Suzina N.E. D., Poulter B., Prinn R.G., Rigby M., Ringeval B., Santini M.,
and Trotsenko Y.A. (2002), Physiological, biochemical, and Schmidt M., Shindell D.T., Simpson I.J., Spahni R., Steele L.P.,
cytological characteristics of a haloalkalitolerant Strode S.A., Sudo K., Szopa S., van der Werf G.R., Voulgarakis
methanotroph grown on methanol, Microbiology, 71(5), A., van Weele M., Weiss R.F., Williams J.E. and Zeng G. (2013),
512-518. Three decades of global methane sources and sinks, Nature
Gilman A., Laurens L.M., Puri A.W., Chu F., Pienkos P.T. and Geoscience, 6(10), 813-823.
Lidstrom M.E. (2015), Bioreactor performance parameters for Kumar S., Bhattacharyya J., Vaidya A., Chakrabarti T., Devotta S.
an industrially-promising methanotroph Methylomicrobium and Akolkar A. (2009), Assessment of the status of municipal
buryatense 5GB1, Microbial Cell Factories, 14(1), 182. solid waste management in metro cities, state capitals, class I
Gogleva A.A., Kaparullina E.N., Doronina N.V. and Trotsenko Y.A. cities, and class II towns in India: an insight, Waste
(2011), Methylobacillus arboreus sp. nov., and Management, 29(2), 883-895.
Methylobacillus gramineus sp. nov., novel non-pigmented Lee H.J., Jeong S.E., Kim P.J., Madsen E.L. and Jeon C.O. (2015),
obligately methylotrophic bacteria associated with plants, High resolution depth distribution of Bacteria, Archaea,
Systematic and Applied Microbiology, 34(7), 477-481. methanotrophs, and methanogens in the bulk and
Gupta S., Choudhary N. and Alappat B.J. Bioreactor landfill for rhizosphere soils of a flooded rice paddy, Frontiers in
MSW Disposal in Delhi. In: Proceedings of the International Microbiology, 6. doi:10.3389/fmicb.2015.00639
Conference on Sustainable Solid Waste Management, Liao J.C., Mi L., Pontrelli S. and Luo S. (2016), Fuelling the future:
Chennai, India, 2007: 474-481 microbial engineering for the production of sustainable
Han B., Chen Y., Abell G., Jiang H., Bodrossy L., Zhao J., Murrell J.C. biofuels, Nat Rev Micro, 14(5), 288-304
and Xing X.H. (2009), Diversity and activity of methanotrophs Mayumi D., Dolfing J., Sakata S., Maeda H., Miyagawa Y., Ikarashi
in alkaline soil from a Chinese coal mine, FEMS Microbiology M., Tamaki H., Takeuchi M., Nakatsu C.H. and Kamagata Y.
Ecology, 70(2), 196-207. (2013), Carbon dioxide concentration dictates alternative
Hansen L.B., Finster K., Fossing H. and Iversen N. (1998), methanogenic pathways in oil reservoirs, Nature
Anaerobic methane oxidation in sulfate depleted sediments: Communications, 13(4). doi:10.1038/ncomms2998.
effects of sulfate and molybdate additions, Aquatic Microbial Murrell J.C., McDonald I.R. and Bourne D.G. (1998), Molecular
Ecology, 14(2), 195-204. methods for the study of methanotroph ecology, FEMS
Hanson R.S. and Hanson T.E. (1996), Methanotrophic bacteria, Microbiology Ecology, 27(2), 103-114.
Microbiological reviews, 60(2), 439-471. Musenze R.S., Fan L., Grinham A., Werner U., Gale D., Udy J. and
Houghton J.T., Ding Y., Griggs D.J., Noguer M., van der Linden P.J., Yuan Z. (2016), Methane dynamics in subtropical freshwater
Dai X., Maskell K. and Johnson C. (2001) Climate change 2001: reservoirs and the mediating microbial communities,
the scientific basis, vol 881. Cambridge university press Biogeochemistry, 128(1), 233-255. doi:10.1007/s10533-016-
Cambridge, 0206-8
Hu P., Chakraborty S., Kumar A., Woolston B., Liu H., Emerson D. Nielsen J., Fussenegger M., Keasling J., Lee S.Y., Liao J.C., Prather
and Stephanopoulos G. (2016), Integrated bioprocess for K. and Palsson B. (2014), Engineering synergy in
conversion of gaseous substrates to liquids, Proceedings of biotechnology, Nat Chem Biol, 10(5), 319-322.
the National Academy of Sciences, 113(14), 3773-3778. doi:10.1038/nchembio.1519
Hwang I.Y., Lee S.H., Choi Y.S., Park S.J., Na J.G., Chang I.S., Kim C., Peralta-Yahya P.P., Zhang F., del Cardayre S.B. and Keasling J.D.
Kim H.C., Kim Y.H., Lee J.W. and Lee E.Y. (2014), Biocatalytic (2012), Microbial engineering for the production of advanced
conversion of methane to methanol as a key step for biofuels, Nature, 488(7411), 320-328.
development of methane-based biorefineries, J. Microbiol. Rawat M. and Ramanathan A. (2011), Assessment of methane flux
Biotechnol., 24, 1597–1605. from municipal solid waste (MSW) landfill areas of Delhi,
Jackson R.B., Canadell J.G., Le Quéré C., Andrew R.M., Korsbakken India, Journal of Environmental Protection, 2(4).
J.I., Peters G.P. and Nakicenovic N. (2015), Reaching peak Sathaye J., Shukla P. and Ravindranath N. (2006), Climate change,
emissions. Nature Climate Change, 6(1), 7-10. sustainable development and India: Global and national
doi:10.1038/nclimate2892 concerns, Current Science-Banglore, 90(3), 314.
Kalyuzhnaya M.G., Yang S., Rozova O.N., Smalley N.E., Clubb J., Schimel J. (2000), Global change: rice, microbes and methane,
Lamb A., Gowda G.A.N., Raftery D., Fu Y., Bringel F., Nature, 403(6768), 375-377.
Vuilleumier S., Beck D.A.C., Trotsenko Y.A., Khmelenina V.N. Sharma S., Bhattacharya S. and Garg A. (2006), Greenhouse gas
and Lidstrom M.E. (2013), Highly efficient methane emissions from India: a perspective, Current Science-
biocatalysis revealed in a methanotrophic bacterium, Nat Banglore, 90(3), 326.
Commun, 4, doi:10.1038/ncomms3785
MOLECULAR DETECTION OF METHYLOTROPHS FROM AN INDIAN LANDFILL SITE 539

Singh J.S. (2011), Methanotrophs: the potential biological sink to

mitigate the global methane load, Current Science, 100(1),
29-30.
Singh V., Mittal A. Toxicity Analysis and Public Health Aspects of
Municipal Landfill Leachate: A Case Study of Okhla Landfill,
Delhi. In: 8th World Wide Workshop for Young Environmental
Scientists WWW-YES 2009: Urban waters: resource or risks?
2-5 June 2009, 2011.
Solomon S. (2007) Climate change 2007-the physical science
basis: Working group I contribution to the fourth assessment
report of the IPCC, vol 4. Cambridge University Press,
Strong P, Xie S. and Clarke W. (2015), Methane as a resource: can
the methanotrophs add value?, Environmental Science and
Technology, 49(7), 4001-4018.
Su Y., Zhang X., Xia F-F, Zhang Q-Q, Kong J-Y, Wang J. and He R.
(2014), Diversity and activity of methanotrophs in landfill
cover soils with and without landfill gas recovery systems,
Systematic and Applied Microbiology, 37(3), 200-207.
Talyan V., Dahiya R. and Sreekrishnan T. (2008), State of municipal
solid waste management in Delhi, the capital of India, Waste
Management, 28(7), 1276-1287.
Talyan V., Dahiya R., Anand S. and Sreekrishnan T. (2007),
Quantification of methane emission from municipal solid
waste disposal in Delhi, Resources, conservation and
recycling, 50(3), 240-259.
Tamura K., Stecher G., Peterson D., Filipski A. and Kumar S. (2013),
MEGA6: Molecular Evolutionary Genetics Analysis Version
6.0., Molecular biology and evolution, 30(12), 2725-2729.
Yadav S., Kundu S., Ghosh S.K. and Maitra S. (2015), Molecular
analysis of methanogen richness in landfill and marshland
targeting 16S rDNA sequences. Archaea (2015):1-9. Article ID
563414, http://dx.doi.org/10.1155/2015/563414
Yadav S., Maitra S., Pal S., Singh N., Gupta S., Ghosh S. and
Sreekishnan T. (2014), Accumulation of Lactic acid during
biodigestion of municipal solid waste leachate and
Identification of Indigenous Lactic Acid Bacteria in leachate,
Journal of Hazardous, Toxic, and Radioactive Waste, 18(4),
04014021.
Yvon-Durocher G., Allen A.P., Bastviken D., Conrad R., Gudasz C.,
St-Pierre A., Thanh-Duc N. and del Giorgio P.A. (2014),
Methane fluxes show consistent temperature dependence
across microbial to ecosystem scales, Nature, 507(7493),
488-491.

View publication stats