Current Microbiology (2021) 78:78–85

https://doi.org/10.1007/s00284-020-02259-x

Exploration of Microbial Diversity of Himalayan Glacier Moraine Soil

Using 16S Amplicon Sequencing and Phospholipid Fatty Acid Analysis
Approaches
Mingma Thundu Sherpa1 · Ishfaq Nabi Najar1 · Sayak Das1 · Nagendra Thakur1

Received: 25 March 2020 / Accepted: 15 October 2020 / Published online: 28 October 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Changme Khangpu glacier is located in the northern district of Sikkim which comes under UNESCO heritage site Kanchen-
junga Biosphere Reserve which is considered as one of the important biological hotspot regions in the Eastern Himalayas.
This is the first report on microbial diversity analysis of moraine soil from one of the unexplored glaciers of Sikkim using
high throughput sequencing platform and phospholipid fatty acids analysis (PLFA). It was found that the 16S amplicon
sequence comprised 362,902 raw sequences with a sequence length of 150 bp and (G + C) content 52%. A total of 156,821
pre-processed reads were clustered into 378 OTUs (operational taxonomic units) comprising 6 bacterial phyla. The top four
dominant phyla based on the 16S amplicon sequences were Proteobacteria (56%), Firmicutes (16%), Actinobacteria (12%),
and Bacteroidetes (8%), respectively. PLFA analysis confirmed the dominance of Gram positive bacteria (72%) followed by
Gram negative bacteria (32%) and the major fatty acids which are present in the moraine soil sample were PUFA (61%), and
18:2ω6,9c (29%). This is the primary study and first of its kind done on moraine soil from glaciers of Sikkim. Based on 16S
amplicon sequencing and PLFA analysis of moraine soil samples from glaciers of Sikkim suggest that this glaciers harbours
rich microbial diversity and thus can have wide industrial and biotechnological potential. Thus, there is an escalating scope
to further study these extreme biomes with respect to their microbial diversity and their functional capabilities.

Introduction of such an extreme environment has got ample attention

A major portion of the earth’s biosphere is cold (<5 °C) and
the glaciers form a larger part of it. Moreover, it has been
known that these portions of the biosphere sustain a broad
range of microbial diversity [1]. These mainly include extre-
mophilic microorganisms such as bacteria, archaea, yeasts,
filamentous fungi, and algae [2]. The Glaciers harbour
microbial communities of great economic importance and
interest to the researchers as their enzymes and metabolites
are being utilized in a wide array of reactions and processes
of industrial application [3]. The microbial diversity analysis
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0028​4-020-02259​-x) contains
supplementary material, which is available to authorized users.
* Nagendra Thakur
nthakur@cus.as.in
1 tats are usually known as psychrophiles. The psychrophiles
Department of Microbiology, School of Life Sciences,
Sikkim University, 6th Mile, Samdur, Tadong,
Gangtok 737102, Sikkim, India
due to their diverse and unique ecology, and geochemistry.
Thus, these habitats provide a great opportunity to explore
novel compounds, metabolites, and their related genes. The
microbial diversity has been observed extensively from cold
habitats all over the world such as that of Arctic and Antarc-
tica regions. Microbial diversity studies have also been done
on ice cores, sampled from different polar and non-polar
glaciers. For instance, Malan and Guliya ice core from the
Tibetan plateau was investigated and the major phylum was
found to be Proteobacteria [4–6]. Similarly, the dominance
of phylum Proteobacteria was also reported from GISP-
2core drilled from the Greenland glacier, Muztag Ata Gla-
cier, China, and Polar ice caps by many researchers [7–10].
However, in context to Himalayan glaciers, the microbial
diversity has been less explored as compared to other cold
habitats. The less exploration might be due to the difficult
topography and less accessibility to these glaciers.
The microorganisms residing in such extreme cold habi-
possess the growth temperatures (minimum, optimum and
maximum) at or below 0, 15 and 20 °C, respectively, whilst

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Exploration of Microbial Diversity of Himalayan Glacier Moraine Soil Using 16S Amplicon…
microorganisms that tolerate cold temperatures with a higher
growth optimum and maximum (above 25 °C) are called
‘psychrotolerant’ [11]. These microbes possess adaptations
towards cold environments by possessing changes in their
membrane composition, cold-active proteins, polyunsatu-
rated lipids, and exopolysaccharides [12]. With these adap-
tations these psychrophiles has been exploited in various
biotechnological, food industries, detergent and fabric indus-
tries, medical and pharmaceutical industries, and bioreme-
diation, etc.
Like other extremophiles, psychrophiles are difficult to
cultivate. As it is well established that approximately 1%
of bacteria on Earth can be readily cultivated by traditional
culture-dependent techniques [13, 14]. However, with the
advent of various culture-independent techniques, the micro-
bial diversity estimation has become easier. Based on the
16S rRNA sequences, most of the bacteria isolated from gla-
cial habitats have close nexus with other terrestrial (perma-
frost soil), and marine environments (sea ice and cold deep
marine sediments) [15, 16]. In contrast, distinctive culture-
free strategies including DGGE, PLFA, and Metagenomics
ponders have uncovered a resulting increment in microbial,
molecular, and ecology studies. A well-known approach
to give an idea about the total biomass and the microbial
diversity present in harsh environments is based on the pres-
ence of various fatty acids present amongst the microor-
ganisms, which is commonly known as phospholipid fatty
acid analysis. This interesting non-culturable technique, i.e.,
phospholipid fatty acid analysis, or PLFA is used for two
decades to characterize microbial communities [17]. It was
initially used to assess the microbial biomass from marine
and estuarine sediments. Later on, it was used to quantify
the viable microbial biomass and identify the biomarkers
for taxonomic evaluation from an environment. The main
significance of PLFA is that it reveals the unique fatty acid
markers present amongst the microbial community network.
On the other hand, other culture-independent methods based
on total environmental DNA estimation and analysis such
as NGS offers a more comprehensive estimation of micro-
bial diversity [18]. Thus, the present study was designed
to investigate the microbial community composition of
Changme Khangpu glacier moraine samples by 16S ampli-
con sequence and PLFA techniques.
Himalayas are one of the tectonically active areas in the
World hosting many alpine glaciers which are the freshwa-
ter sources for billions of people in South East Asia. The
Changme Khangpu glacier is one of the less explored gla-
ciers in Sikkim, India. The mean temperature and pH of the
glacier are usually −45 °C and 7, respectively [3]. Till date,
available report on this glacier is based on culturable bacte-
rial diversity analysis of glacier accumulation zone only [3,
19, 20]. However, no data are present on the moraine area of
this glacier. Thus, the bacterial diversity based on amplicon
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
79
sequence of V3 and V4 region of bacterial 16S rRNA gene
was amplified using two primers (16S_V3-F = 5′-TCG​TCG​
GCA​GCG​TCA​GAT​GTG​ATA​AGAG-ACA​GCC​TAC​GGG​
NGGC ​ WCA​ G -3′; 16S_V3-R = 5′-GTC ​ T CG​ T GG ​ G CT​
CGG​AGA​TGTG-TAT​AAG​AGA​CAG​GAC​TAC​HVGGG​
TAT​CTA​ATC​C-3′) and phospholipids fatty acid analysis
were carried out from Changme Khangpu glacier moraine
of Sikkim.
Materials and Methods
Sample Collection
Lateral and supra glacier samples were collected in the
month of September 2017. A total of seven debris based
samples 30–40  g each were collected aseptically from
moraines. The samples were collected in 50 ml Falcon tubes
by laboratory spoons and kept at 4 °C during transportation
to the laboratory within 24 h. For each sampling site, the
GPS coordinates were assessed through a GPS MAP 78S,
an automated Global Positioning device. The coordinates
were processed through the programmed software Google
Earth for mapping.
Physicochemical Analysis of Changme Khangpu
Moraine Sample
Physical parameters were measured with the help of U-50
Multi-parameter water quality checker, Horiba, Japan and
Temperature-Infrared Thermometer Non-Contact Gun Laser
IR Point Digital LCD Temperature, Taiwan, China. Chemi-
cal parameters were analysed with the help of Multiparam-
eter water testing kit system-AquaLab Himedia, Mumbai,
India.
Phospholipids Fatty Acid Analysis (PLFA)
For PLFA analysis, the phospholipids were extracted from
moraine sample according to the standard protocol [12, 21]
and were analysed using Sherlock-MIDI identification sys-
tem. The calibrated standards were used by the microbial
identification system (MIDI) for annotation of generated
phospholipids peaks.
DNA Extraction
Environment DNA was extracted from moraine samples of
the glacier using a DNeasy Power Water Kit (MO BIO Labo-
ratories, Carlsbad, CA, USA). DNA was quantified using
Qubit Fluorometer (Thermofisher Scientific, USA), with a
detection limit of 10–100 ng μL−1 and the quality of the
DNA was checked on 0.8% Agarose gel.
13

80 M. T. Sherpa et al.

Sequencing and Bioinformatics Results

The V3 and V4 region of the 16S rRNA gene was amplified
using two primers (16SV3F = 5′TCG​TCG​GCA​GCG​TCA​
GAT ​ G TG​ ATA ​ AGA​ G AC​ AGC ​ C TA​ C GGGNGGC ​ WGC​
AG); (16SV3R = 5′GTC​TCG​TGG​GCT​CGG​AGA​TGT​GTA​
TAAG ​ AGA ​ CAG ​ GAC
​ TACHVGGGT ​ ATC​ TAA
​ TCC​ 3′) [22].
The amplicon libraries were prepared using the Nextera XT
Index Kit (Illumina Inc.), in accordance with 16S metagen-
omic sequencing library preparation protocol [23]. The
amplicon library was purified with AMPure XP beads. The
amplified library was checked by Bioanalyzer 2100 (Agilent
Technologies) using High Sensitivity (HS) DNA chips and
concentration was quantified by Qubit fluorometer. Based
on the data obtained from the Qubit fluorometer and the
bioanalyzer, 500 uL of the 10 pM library was loaded into
MiSeq cartridge for cluster generation and sequencing. A
paired-end sequencing method was used. After the sequenc-
ing, high quality 16S amplicon sequence reads were trimmed
to remove the barcode and adaptor sequences [24].
The adapter trimmed sequence was subjected to pre-pro-
cessing for De-replication, Singleton removal, OTU Clus-
tering, Chimera filtering with SolexaQA. Sequences with
Phred score lower than 20 and ambiguous bases having
primer mismatch and low read length 100 bp were removed.
Annotation and normalization of the operational taxonomic
unit (OTU) was done using UPARSE OTU clustering and
QIIME at 97% similarity [25]. For normalization, an inbuilt
script, as well as METAGEN assist, was used. The resulting
representative OTU was aligned and given taxonomic class-
ing using the Green genes database (https​://green​genes​.lbl.
gov). The output of this workflow is a classification of reads
at several taxonomic levels: kingdom, phylum, class, order,
family, genus, and species. Sequences without homologous
pair were classified as unknown.
Mapping and Assessment of Physicochemical
Parameters of Changme Khangpu Glacier Moraine
The geological coordinates of the Moraine area of the
Changme Khangpu glacier moraine were measured using
the GPS MAP 78S system. The coordinates were found
as 27°55″ 36.03 N and 88°41″ 08.37 E (Supplementary
Fig. 1). The elevation was found to be 15,887 feet above
the sea level. The temperature was found to be −15 °C
and the pH was found to be 7.3. Moreover, the oxida-
tion–reduction potential (ORP) was found to be 218 mv
with dissolved oxygen (DO) as 8.98 mgL −1 as shown in
Supplementary Tables 1 and 2. Chemical parameters such
as fluoride were (0.5 mgL −1), nitrate (10 mgL −1), iron
(0.3 ppm), chloride (20 ppm), turbidity (>25 NTU), and
free chlorine (0.2 ppm). Using google map software and
applying geological coordinates the map was created as
shown in Supplementary Fig. 1.
Phospholipid Fatty Acid Analysis (PLFA Analysis)
PLFA analysis with the Sherlock PLFA tool was utilized to
define the community structure of the moraine soil sample
of Changme Khangpu glacier. The results have shown the
dominance of Gram-positive bacteria (47.20%) followed
by Gram-negative bacteria (4.22%) (Fig. 1; Supplemen-
tary Table 3). The abundance of various fatty acids was
also investigated and it was demonstrated that the straight-
chain fatty acids, branched-chain fatty acids, and PUFA
were abundant as shown in Supplementary Table 4.

Fig. 1  Community structure PLFA based diversity

based on PLFA analysis. For
PLFA analysis, the phospholip- 50.00%
ids were extracted from moraine 45.00%
samples and were analysed
using Sherlock-MIDI identifica- 40.00%
Abundance in %

tion system 35.00%

30.00%
25.00%
20.00%
15.00%
10.00%
5.00%
0.00%
Gram Positive Gram Negative Anaerobes Fungi

13

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Exploration of Microbial Diversity of Himalayan Glacier Moraine Soil Using 16S Amplicon… 81

16S Amplicon Sequencing
16S amplicon sequence analysis showed the dominance
of phylum Proteobacteria (56%) with the major classes as
Beta-Proteobacteria and Gamma-Proteobacteria. After
Proteobacteria the phylum Firmicutes (16%) predominant
followed by Actinobacteria (12%), and Bacteroidetes (8%)
(Fig. 2). The analysis at the genus level showed the domi-
nance of Bifidobacterium, Herminiimonas, Noviherbaspiril-
lum, and Pseudomonas as shown in Fig. 3. Further analysis
at the species level, revealed the presence of 22 bacterial
species with dominance of Herminiimonas saxobsid-
ens, Noviherbaspirillum suwonense and unknown species
(Fig. 3). The presence of unknown species (8%) strongly
suggested that Changme Khangpu glacier moraine harbour
the wealth of many novel and uncultivable bacteria.
Diversity indices were calculated and it was found that
there was diversity in the studied ecosystem. The Shannon-H
index value was 3.053 whereas, Fisher Alpha and Chao-1
was found to be 8.719 and 22, respectively. The rarefaction

Abundance of major phyla Abundance of major classes

60% 30%

Abundance in %
50%
Abundance in %

20%
40%

30% 10%
20%

10% 0%
Betaproteobacteria Gammaproteobacteria
0% Actinobacteria Alphaproteobacteria
Firmicutes Actinobacteria Bacilli Clostridia
Bacteroidetes Planctomycetes Cytophagia Negativicutes
Thaumarchaeota Proteobacteria Planctomycetia Sphingobacteria

Fig. 2  Distribution of various microbial phyla and class based on V4 region of 16S rRNA) metagenomic sequencing library prepara-
metagenomic study. The amplicon libraries were prepared using the tion protocol and loaded into MiSeq cartridge for cluster generation
Nextera XT Index Kit (Illumina Inc.), in accordance with 16S (V3– and sequencing

Abundance of major genus Abundance of major species

9%
9%
8%
8%
7%
Abundance in %

7% 6%
Abundance in %

6% 5%
4%
5%
3%
4%
2%
3% 1%
2% 0%

1%

0%

Bifidobacterium Herminiimonas
Noviherbaspirillum Pseudomonas
Acinetobacter Escherichia/Shigella
Faecalibacterium Hymenobacter
Lactobacillus

Fig. 3  Abundance of major bacterial genus and species

13

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82
curve was plotted with the number of individuals on the
x-axis against the percent taxa on the y-axis (confidence
interval of 95%). The rarefaction curve is given in Fig. 4,
signifies that the curve has reached a horizontal asymptote,
thus can be inferred that the value of rarefaction is a good
estimate of the value that would be obtained if every indi-
vidual was observed at least once. Rarefaction countenances
the calculation of species richness in a sample. The plateau
formed on the plot also signifies that other rare species can
be identified by intensifying the sampling as shown in Fig. 4.
Discussion
Mountain glaciers as in the Himalayan regions, Alps,
and Adens are chiefly constrained by topography and
are mainly of the valley type glaciers [26]. As compared
to Polar glaciers, mountain glaciers are host to diverse
microbial communities carrying out important ecosystem
processes [27]. In contrast to polar ice sheets, valley type
mountain glaciers are often characterized by steep eleva-
tion gradients, high levels of hydrological connectivity
from the surface to base via crevices, fractures, and mou-
lins, and greater snow accumulation [27, 28]. Himalayan
glacier ecosystems contain diverse habitats that can be
divided into three main zones: accumulation zone (the
zone where snow and ice accumulate by snowfall, hail,
drifting snow, avalanche, and rainfall that freezes); abla-
tion zone (loss of ice takes place through the process of
melting, evaporation, calving, and forms supra-glacial lake
with moraines); and Snout (the terminus of the glacier
and is the part of ablation zone). The major portion of
the ice of the glaciers on earth is covered by Antarctica,
Greenland ice caps sheet, polar and non-polar glaciers
Fig. 4  Rarefaction curve
13
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M. T. Sherpa et al.
that cover roughly 10–12% of earth’s land and generate
approximately 70–75% of perennial freshwater on earth.
Himalayan glaciers are the major source of freshwater for
millions of peoples on the earth [29]. Changme Khangpu
glacier is located in the North district of Sikkim, India.
This glacier originated from the southern slope of Mt.
Gurudongmar peak and is also a debris-covered valley
type glacier. The melt-water of this glacier feds into the
Lachung river which is the main tributaries of Tista river.
In glacier habitats, microbes (bacteria, archaea, viruses,
fungi, and algae) are carried and deposited on the surface of
snow by the wind from different places. Glacier ice entraps
and preserve the aerosol dust particles containing biological
materials. The eroded materials in wind varies in content
and quality depending on the topology, local and global
environmental conditions create different local snow eco-
logical system and thus represents a dynamic nutrient and
microbial reservoirs in glaciers [30].
The microbial diversity studies from polar and non-polar
environments have been also carried out. These habitats have
found to be dominated by phylum Proteobacteria, Cyano-
bacteria, Actinobacteria, Bacteroidetes and Firmicutes.
Similarly, Beta-proteobacteria is the dominant class detected
by many researchers in the glaciers [31–35].
Our study is the first of kind from Changme Khang gla-
cier moraine samples and this finding provides a description
of the microbial diversity especially from the moraine sur-
face of the Ablation zone. The dominant phylogenetic group
established by metagenomic approaches was phylum Pro-
teobacteria (56%) followed by Firmicutes (16%), Actinobac-
teria (12%), and Bacteriodetes (8%). Most proteobacterial
sequences were assigned to the class Beta-proteobacteria,
which accounted for almost half (24%) of all Proteobacteria,
with Alpha- proteobacteria (12%) and other classes account-
ing for the remainder. This corresponded to other studies
of glacier ice [16], subglacial habitats [36], and mountain
snow [37].
In addition, a large part of the sequences grouped into
the gram-positive bacteria mainly Actinobacteria. It has
been revealed that Actinobacteria have a higher capacity
to survive in cryo-habitats than low GC gram-positive and
gram-negative bacteria [38]. This might be probably due
to the ability of Actinobacteria to develop resting forms
with low metabolic activity. All other bacterial phyla were
represented by <5% of the classified sequences. Thus, the
phylum Proteobacteria followed by Firmicutes, and Act-
inobacteria represented the predominant in glacier moraine
from the Changme Khangpu glacier, North Sikkim, India.
These types of phyla were also reported from several other
polar and non-polar glaciers [36, 39], other cold habitats
such as glacial melt-water [16, 40], snow [37], and Antarctic
lakes [41]. Thus, these results suggest the perception that the
similar types of phylotypes might exist in geographically
Exploration of Microbial Diversity of Himalayan Glacier Moraine Soil Using 16S Amplicon…
Fig. 5  Principle component
analysis
diverse cold habitats because of similar types of strategies
for survival and remaining active at low temperature [6].
The principal component analysis (PCA) was performed
to check the correlation amongst various variables such as
between various phyla and physicochemical parameters. The
PC1 was showing the highest percentage of 97.94%, thus
was considered (Fig. 5). The biplot suggested that tempera-
ture is correlated to various phyla such as Actinobacteria,
Firmicutes, and Bacteroidetes, etc. these results were also
supported by the findings that Actinobacteria have a higher
capacity to survive at cold temperatures [38].
However, Proteobacteria which is showing a negative
correlation with temperature as shown in biplot has been
also supported by the fact that Proteobacteria tend to
predominate in moderate temperatures [5]. On the other
hand, Proteobacteria have shown a positive correlation with
pH. These findings reveal that glaciers are the hot spots for
research to correlate various parameters or processes such
as climate change, water depletion, and other geological and
microbiological aspects.
Conclusion
To best of our knowledge, this is the first report of the
culture-independent bacterial diversity analysis from gla-
cier moraine samples from Sikkim. These results broad-
ened the view and supported the idea that Himalayan
glacier moraine surface harbour rich microbial diversity.
The 16S amplicon sequencing showed that phylum Proteo-
bacteria predominate with 56% (in particular to the class
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83
Beta-Proteobacteria ~24%) whereas PLFA analysis sug-
gested the predominance of Gram-positive bacteria. The
microbial study of this glacier will be a benchmark data
for future studies and also there might be more exploration
of some known or novel bacteria that might be useful to
develop the desire cryoenzymes in near future for biotech-
nological or industrial purposes. The importance of these
enzymes is their flexible structure which reimburses at cryo
conditions.
Acknowledgements  The authors would like to thank the Department
of Forest, Govt. of Sikkim for kind permission and Department of
Microbiology, Sikkim University for laboratory space.
Compliance with Ethical Standards 
Conflict of interest  The authors declare that there are no competing
interests.
Nucleotide sequence accession number  Metagenome sequence data
are available at NCBI Accession no. SRP165759 (direct link to deposi-
tion data: https​://www.ncbi.nlm.nih.gov/sra/SRX48​87442​).
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