draft - university of toronto t-space · 13 email: [email protected] 14 molecular and...

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Diverse culturable bacterial communities with cellulolytic

potential revealed from pristine habitat in Indian trans-

Himalaya

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2017-0754.R2

Manuscript Type: Article

Date Submitted by the Author: 14-Apr-2018

Complete List of Authors: Thakur, Vikas; Institute of Himalayan Bioresource Technology CSIR

Kumar, Vijay; Institute of Himalayan Bioresource Technology CSIR Kumar, Sanjay; Institute of Himalayan Bioresource Technology CSIR Singh, Dharam; Institute of Himalayan Bioresource Technology CSIR,

Keyword: Cellulose degradation, Psychrotrophs, Thermophiles, High altitude regions, Western HImalaya

Is the invited manuscript for consideration in a Special

Issue? : Not applicable (regular submission)

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Title: Diverse culturable bacterial communities with cellulolytic potential revealed from pristine 1

habitat in Indian trans-Himalaya 2

Authors: Vikas Thakur1,2, Vijay Kumar1, Sanjay Kumar1, and Dharam Singh1,* 3

Author’s Affiliation: 4

1Biotechnology Division, CSIR- Institute of Himalayan Bioresource Technology, Post Box No. 6, 5

Palampur-176 061, Himachal Pradesh, India. 6

2Academy of Scientific and Innovative Research (AcSIR), CSIR- Institute of Himalayan Bioresource 7

Technology, Palampur, Himachal Pradesh, India 8

Emails: 9

[email protected] (VT), [email protected] (VK), [email protected] (SK), 10

[email protected] (DS) 11

*Corresponding author: 12

Email: [email protected] 13

Molecular and Microbial Genetics Lab, Biotechnology Division, CSIR-Institute of Himalayan 14

Bioresource Technology, Palampur, Himachal Pradesh, INDIA-176061 15

Phone: +91-1894-233339, ext. 422 16

Fax: + 91-1894-230433 17

Running Title: Efficient cellulolytic bacteria from Indian trans-Himalaya 18

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Abstract 1

Pangi-Chamba Himalaya (PCH) region is very pristine, unique and virgin niche for bioresource 2

exploration. In the current study, for the first time, the bacterial diversity of this region for potential 3

cellulose degrader was investigated. A total of 454 pure bacterial isolates were obtained from diverse 4

sites in PCH region and 111 isolates were further selected for 16S rDNA characterization based on 5

ARDRA grouping. Identified bacteria belongs to twenty-eight genera representing four phyla namely 6

Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. Pseudomonas was most abundant 7

genera followed by Bacillus, Geobacillus, Arthrobacter, Paenibacillus, and Flavobacterium. In 8

addition, 6 putative novel bacteria (based on 16S rDNA sequence similarity) and thermophiles from 9

non-thermogenic sites were also reported for the first time. Screening for cellulose degradation ability 10

on carboxymethyl cellulose (CMC) plates had revealed 70.92% of cellulolytic bacteria. Current study 11

reports diverse genera (Arthrobacter, Paenibacillus, Chryseobacterium, Pedobacter, Streptomyces, 12

Agromyces, Flavobacterium, and Pseudomonas), high cellulose hydrolysis zone, and wide pH and 13

temperature functional cellulolytic bacteria hitherto reported in the literature. Diverse bacterial genera 14

with high cellulolytic activity in broad pH and temperature range provide opportunity to develop a 15

bioprocess for efficient pretreatment of lignocellulosic biomass, which is currently being investigated. 16

Keywords Cellulose degradation; Psychrotrophs; Thermophiles; High altitude regions; Western 17

Himalaya 18

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Introduction 1

Microorganisms are ubiquitous in all environmental niches such as lakes, hot springs, 2

glaciers, deep oceans and high mountains. The general perception of a cold ecosystem is that of the 3

polar region. However, high altitude mountains ranges such as the Himalayas also play a significant 4

role as the cold pockets of the biosphere and inhabited by the diverse array of microorganisms (Stres et 5

al. 2013). In recent years, high altitude and colder regions have gained much attention from several 6

researchers for microbial exploration (Shivaji et al. 2011; Lutz et al. 2015; Venkatachalam et al. 2015; 7

Ambrosini et al. 2016). The distinctive habitat features of multiple stress conditions of fluctuating 8

temperature, low oxygen, and atmospheric pressure, high wind velocity and UV radiations, water and 9

nutrients scarcity create a biological resource with novelty and unique properties (Carpenter et al. 10

2000; Albarracin et al. 2012; Lutz et al. 2015; Ambrosini et al. 2016). In the past, attempts have been 11

made to explore the bacterial diversity based on culture-dependent and metagenomic approaches and 12

reported various bacterial phyla such as Proteobacteria, Fermicutes, Actinobacteria, Acidobacteria 13

and Bacteroidetes from Himalayan regions (Gangwar et al. 2009; Pradhan et al. 2010; Shivaji et al. 14

2011; Srinivas et al. 2011; Gupta et al. 2015). With the increasing interests in the Himalayan regions, 15

more efforts and intensive study are needed to understand the highly dynamic microflora ′and to 16

harness their biotechnological potentials. 17

Pangi-Chamba region in trans-Himalaya of Himachal Pradesh (31°58'92'' N, 78°27'75'' E) was 18

contemplated to be our study area due to unpredictable environmental conditions, fluctuating 19

temperature, unexplored micro-flora, and minimum human inference. This region has altitude variation 20

from 2000 to 5000 meters from mean sea level and provides a rare blend of distinct topographical 21

terrenes constituting glaciers, lakes, water streams, alpine regions, rocky mountain and forests (Fig. 1). 22

Thus, provides unique opportunities for studying microbial diversity and biological alterations in the 23

genome of microbes that bestowed them with the ability to synthesize novel proteins/enzymes or 24

molecules to survive in the harsh environmental conditions. Microorganisms thriving in such harsh 25

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environments can be excellent source of enzymes of industrial applications (Van den Burg 2003; 1

Elleuche et al. 2014; Sarmiento et al. 2015; Santiago et al. 2016). The cold active bacterial enzymes 2

have low chemical reaction rates, however, to compensate proteins are evolved with high catalytic 3

efficiency by augmenting flexibility in protein structures (Siddiqui and Cavicchioli 2006). The unique 4

features of cold active enzymes such as high catalytic efficiency, high substrate affinity and stability in 5

the broad range of pH and temperature (Siddiqui and Cavicchioli 2006; Santiago et al. 2016; 6

Cavicchioli et al. 2011) are assets to the industry. Several studies revealed the presence of enzymes 7

like phytase, lipase, amylase, protease, cellulase, etc. from microbes of Himalayan regions (Gangwar 8

et al. 2009; Pradhan et al. 2010; Salwan et al. 2010; Shivaji et al. 2011; Bhat et al. 2013; Sahay et al. 9

2013; Venkatachalam et al. 2015). Especially, cellulases are a group of glycoside hydrolases that 10

catalyses the breakdown of β-1, 4 linkages in cellulosic biomass to form simpler sugars. It’s been over 11

80 years since the first report of cellulose degrading potential of Trichoderma reesei strain from 12

Solomon Islands (Bischof et al. 2016), but still there is a dire need of a reliable cellulase system which 13

can provide efficient and complete transformation of the cellulosic substrate into simple sugars. 14

Cellulose biomass is abundant in nature and can be utilized in a number of applications using cellulose 15

degrading enzymes at ambient reaction conditions (Cragg et al. 2015; Payne et al. 2015). There are 16

number of predicaments with cellulosic biomass bioconversion like incomplete degradation due to 17

limited substrate access, narrow pH and temperature range, low catalytic activity, product inhibition 18

and stability issues (Arantes et al. 2010; Balan 2014; Jordan and Theegla 2014; Jeoh et al. 2017). 19

Cellulase enzyme was chosen for current study due to its applicability and requisite in various 20

industries i.e. textiles, pulp, food, detergent, biopolishing, bioethanol and chemical industry (Kuhad et 21

al. 2011; Hasunuma et al. 2013; Juturu et al. 2014; Sharma et al. 2016; Valdivia et al. 2016; Jeoh et al. 22

2017). Moreover, natural decay of lignocellulosic biomass in the high altitude regions of water stressed 23

niches creates favorable conditions for bacterial species with high cellulase activity. Therefore, 24

realizing the unprecedented opportunities of Himalayan bioresources and industrial applications of 25

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cellulases, the present study was aimed to identify and harness bacterial diversity of pristine PCH 1

region for efficient, stable, wide temperature and pH operative cellulase enzyme. 2

Materials and Methods 3

Study site description and sampling 4

Pangi Valley (33°00'20.2"N, 76°14'23.2"E) is one of the rugged and remotest areas in Himachal 5

Pradesh covering 1,601 square kilometers and elevation 8789 feet (Fig. 1). Samples were collected 6

from different locations such as Sural, Killar, Hudan and Saichu Valleys located at an altitude range of 7

7,000 feet (2,100 m) to 11,000 feet (3,400 m) above sea level. In addition, the Sach Pass, summer 8

gateway to Pangi Valley was the highest point for sampling with an altitude of 14,500 feet (4,400 m). 9

The temperature ranges from as low as -40°C in winters to 30°C in summer. Besides, there is drastic 10

fluctuation in day and night temperature. For reconnaissance of microflora in these ranges, 33 samples 11

consisting 18 soils, 6 compost and 9 lake water samples were collected from different locations in 12

Pangi. Soil samples collected from 3-5 inches depth using sterile trowel in bags and polypropylene cap 13

bottles. Samples were brought to the laboratory in either liquid nitrogen (-196°C) or ice cooled packs 14

and stored in 4°C cold room for further use. 15

Bacterial isolation 16

Bacterial isolation was carried out at 4°C, 10°C, 20°C, 37°C and 50°C using four different media viz., 17

Nutrient agar, R2A, Antarctic bacterial medium (ABM) and modified Thermus media containing 18

peptone 8 g/L, yeast extract 4 g/L, NaCl 2 g/L and agar 2%. Dilutions were prepared for both soil and 19

compost samples, while water samples were spread on plates directly. Further, the plates were 20

incubated at different temperatures of 4, 10, 20, 28 and 37°C. All the samples were diluted up to 10-5 21

while at 50°C incubation; samples were diluted to 10-2 in 0.85% saline solution. Plates were incubated 22

for different periods until visually distinct colonies appeared at 4°C, 10°C, 20°C, 37°C and 50°C after 23

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14, 10, 3, 2 and 2 days, respectively. Colonies were counted manually by plate count method. By 1

phenotypic characteristics (color, size, exo-polysaccharide), different bacterial colonies were selected 2

and re-purified using streak plate method. After sub-culturing the bacterial isolates were preserved in 3

25% glycerol and stored at -80°C. 4

Partial gene sequencing of 16S rDNA 5

DNA isolation was performed by the heat-chill method of lysate preparation. Pure isolated colonies 6

were inoculated in 70µl Milli-Q water, vortexed and kept in -80°C for 15 min. Samples were then 7

immediately subjected to 95°C in Thermal cycler for heat shock. This heat-chill method ruptures the 8

bacterial cell wall, releasing all cellular contents including genomic DNA. Samples centrifuged at 9

11000 g for 10 min, and the supernatant was collected. This supernatant containing genomic DNA 10

used as a template for 16S rRNA gene amplification using two universal primers 27F (5'-11

AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'–TACGGYTACCTTGTTACGACTT-3'). 12

Amplified product visualized on 1.2% agarose gel. PCR product was then subjected to ARDRA 13

(Amplified Ribosomal DNA Restriction Analysis) with different restriction enzymes (Taq1-T/CGA, 14

HaeIII -GG/CC, HhaI-GCG/C, MspI- C/CGG) and resolved on 3% agarose gel. Unique phylotypes 15

were selected based on their banding pattern observed in ARDRA. Purification of PCR products of 16

selected phylotypes was carried out using ExoSAP-IT (Thermo Fisher Scientific) and subjected to 16S 17

rDNA sequencing via ABI 3130XL Genetic Analyzer. 18

Phylogenetic analysis 19

The sequences were compared to sequences available in GenBank using BLAST (Basic Local 20

Alignment Search Tool), aligned using MEGA6.06 software and identified using Ez-Taxon server. In 21

the end, a phylogenetic tree was constructed using BioEdit and MEGA6.06 software employing the 22

neighbor-joining method based on Jukes-Cantor model with 1000 replicates of bootstrap. 23

Qualitative estimation of cellulolytic activities 24

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Qualitative screening for cellulase activity was carried out for purified bacterial isolates using Grams 1

iodine method (Kasana et al. 2008). Isolates were screened for cellulase activity on modified CMC 2

media containing K2HPO4 0.25g, MgSO4 0.125g, NaNO3 0.5g, peptone 0.05g, KCl 0.125g, carboxy 3

methyl cellulose (CMC) 1.25 g and agar 2% in 250ml distilled water (Singh et al. 2013). Single 4

isolated bacterial colonies were spotted on cellulase screening media plates and incubated for 36 to 72 5

h depending on their growth. Plates then flooded with Gram’s Iodine solution (Iodine 1g, potassium 6

iodide 2g in 300mL dH2O), kept for 10 min and washed with 1N NaCl. Clear hydrolysis zones were 7

observed indicating the presence of cellulolytic activities. Zone ratio and HC/Cellulolytic index values 8

were obtained according to the formulas described elsewhere (Ferbiyanto et al. 2015). 9

Results 10

Isolation of bacteria from diverse soil and lake water samples of PCH region 11

Four hundred and fifty-four bacterial culture were isolated and subcultured on nutrient agar, R2A, 12

ABM and Thermus media from the soils, water and compost samples collected from PCH region. 13

Based upon their optimum growth carried out at 4°C, 10°C, 20°C, 37°C and 50°C, 355 isolates were 14

psychrotrophic, 35 mesophiles and 64 were of thermophilic nature. Most of the bacterial isolates can 15

grow well in pH range from 5 to 10 and can tolerate NaCl concentration up to 2%. The 454 pure 16

cultures were subjected to 16S rRNA gene amplification followed by restriction digestion analysis 17

(ARDRA), grouped into 111 phylotypes (69 psychrotrophs, 9 mesophiles and 33 thermophiles). 18

Further, these phylotypes were sequenced by Sanger sequencing method and their nucleotide 19

sequences were submitted to NCBI-GenBank database (Table S1). 20

Diversity of culturable bacteria from PCH region under selected laboratory conditions 21

The culturable bacterial diversity in PCH region belonged to 28 different genera representing four 22

phyla; Firmicutes (45%), Proteobacteria (34%), Bacteroidetes (11%) and Actinobacteria (10%) (Fig. 23

2). Phylogenetic analysis revealed the abundance of Firmicutes with fifty isolates belonging to nine 24

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genera namely Bacillus, Staphylococcus, Saccharococcus, Geobacillus, Laceyella, 1

Thermoactinomyces, Exiguobacterium, Brevibacillus, and Paenibacillus. Proteobacteria was second 2

most abundant phylum with γ-proteobacteria (27%), β-proteobacteria (5%) and α-proteobacteria 3

(2%). This phylum is also found to be the most diverse with twelve genera i.e. Sphingomonas, 4

Caulobacter, Janthinobacterium, Duganella, Burkholderia, Delftia, Stenotrophomonas, Citrobacter, 5

Lelliottia, Enterobacter, Acinetobacter, and Pseudomonas. Besides, four genera belonging to phylum 6

Actinobacteria namely Streptomyces, Agromyces, Microbacterium, Arthrobacter, and three from 7

Bacteroidetes i.e. Pedobacter, Flavobacterium, Chryseobacterium were also observed (Fig. 3). Among 8

the Genus, Pseudomonas was most dominant (22 isolates) followed by Bacillus (21 isolates), 9

Geobacillus (16 isolates), Arthrobacter (7 isolates), Paenibacillus, Flavobacterium, and 10

Chryseobacterium each with five isolates. A phylogenetic tree based on neighbor-joining method 11

showed the diverse genera of four phyla of PCH region (Fig. 4). 12

Identification of putative novel isolates from PCH region 13

Among the identified culturable bacteria, six bacterial isolates having 16S rDNA percentage similarity 14

less than 98.6% with reference type strains in EzTaxon (Kim et al. 2014) are considered to be putative 15

novel (Table 1). Among them, two isolates PCH 8 and PCH 35 belonged to genus Paenibacillus 16

having 96.9 and 97.95% similarity with type strain Paenibacillus amylolyticus NRRL NRS-290(T) and 17

Paenibacillus endophyticus PECAE04(T), respectively. Two isolates PCH 22 and PCH 74 belonged to 18

genus Flavobacterium and matched with nearest neighbor Flavobacterium hercynium WB 4.2-33(T) 19

(98.57%) and Flavobacterium aquidurense WB-1.1.56(T) (98.46%). PCH 117 had 97.14% similarity 20

with Saccharococcus thermophilus ATCC 43125 (T), and PCH 34 found Acinetobacter schindleri CIP 21

107287(T) as the closest match with 97.86%. The strain PCH8 has already been submitted and 22

assigned Microbial Type Culture Collection (MTCC, CSIR-IMTECH Chandigarh, India) accession 23

number 12597 and other bacterial isolates are in process of submission. In addition to above six 24

putative novel isolates, five more isolates were found with a similarity to a type strains between 98.7 to 25

99%. These five isolates were PCH 19, 37, 42, 62 and 75 having closest match with Flavobacterium 26

hydatis DSM 2063(T) (98.86%), Flavobacterium hydatis DSM 2063(T) (98.75%), Caulobacter 27

henricii ATCC 15253(T) (98.98%), Microbacterium kyungheense THG-C26(T) (98.98%) and 28

Chryseobacterium piperi CTM(T) (98.78%), respectively. 29

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Identification of thermophiles from PCH region 1

In this study, thermophilic bacteria were successfully isolated from PCH region, a non hot-spring or 2

non-thermogenic area. Among the 33 thermophilic bacteria (Serial number 79-111 of Table S1), 16 3

isolates belonged to Geobacillus (~15% of total sequenced isolates). The other 17 isolates represent 4

various bacterial genera which includes Bacillus, Streptomyces, Brevibacillus, Thermoactinomyces, 5

Laceyella, and Saccharococcus. These bacterial isolates can grow at 55°C but fail to grow below 37°C. 6

Importantly, above six genera excluding Geobacillus were isolated for the first time as thermophilic 7

bacteria from the colder regions in general and specifically from the Himalayas. 8

Qualitative screening of bacterial isolates for cellulase activity 9

Cellulases are a diverse group of enzymes that break down cellulose to form simple sugars which 10

have various applications in the industry. In the present study, total 322 isolates (~71%) were found 11

positive for cellulase activity by CMC plate screening. Among the molecular characterized 111 12

bacterial isolates representing 454 total isolates, 67 have found positive for cellulolytic activity (Table 13

S2). Firmicutes are reported with the higher number of cellulase positive followed by Proteobacteria, 14

Bacteroidetes, and Actinobacteria (Fig. 5). On comparing cellulase positive to overall culturable 15

diversity within the phylum, Bacteroidetes possess highest percentage (92%) for cellulolytic ability 16

followed by Actinobacteria (73%) trailing by Firmicutes (66%) and Proteobacteria (37%). 17

Proteobacteria notably represent the highest diversity at genus level, as 14 isolates belonging to five 18

genera (~41%) were found positive for cellulase activity in plate assay. In overall diversity, the most 19

prominent genera for cellulolytic capacity belonged to Bacillus, Geobacillus, Paenibacillus 20

(Firmicutes), Pseudomonas (Proteobacteria), Arthrobacter, Streptomyces (Actinobacteria), 21

Pedobacter, Flavobacterium and Chryseobacterium (Bacteroidetes). The isolates showing hydrolytic 22

zones in CMC agar plates were selected (Fig. 6) and their HC values were calculated (Table S2). 23

Flavobacterium sp. PCH 74 had shown highest zone ratio of 9 followed by Flavobacterium sp. PCH 24

81 (6.25), Geobacillus sp. PCH113 (6), Paenibacillus sp. PCH82 (5.5), Bacillus sp. PCH106 and 25

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Chryseobacterium sp. PCH 75 (each had 5), whereas, Agromyces sp. PCH26, Paenibacillus sp. 1

PCH60 and Exiguobacterium sp. PCH83 had a zone ratio of 4.5, 4.5 and 4.33, respectively (Table 2). 2

Discussion 3

Unique geology, climatology, and ecology of Himalayan regions fascinated many researchers for the 4

bioprospection. Earlier, extreme west Himalayan regions have been explored for microbial diversity 5

using culture-dependent and independent methods (Gangwar et al. 2009; Pradhan et al. 2010; Shivaji 6

et al. 2011; Srinivas et al. 2011; Gupta et al. 2015; Venkatachalam et al. 2015; Ambrosini et al. 2016). 7

However, PCH region (west Himalaya) is still unexplored for bioresource discovery and their 8

utilization due to inaccessibility and tough terrains. In fact, this region is located between two 9

mountain ranges namely Zanskar and Pir Panjal in the western Himalaya (Fig. 1) making its habitat 10

unique and free of anthropogenic disturbance. Further, harsh environmental conditions and varied 11

temperature during winter and summer season earned this region a status of gold mine for the 12

identification of novel bacteria and enzymes or molecules of industrial importance. 13

Cellulases are the key enzymes having various applications in pulp and paper, food, textiles, 14

detergents and bioethanol industry. Global market for enzymes has been estimated to reach 17.5 USD 15

billion by 2024 with a CGAR of 9.2% from 2016 to 2024 (Enzyme market, 2016). With huge market 16

potential and increasing global demand, PCH region was explored for its culturable bacterial diversity 17

with a focus to find the cellulose hydrolyzing bacteria. In this investigation, about 71% (322/454) of 18

total isolated bacteria were screened positive for cellulase activity in plate-based assay (Table S1). The 19

identified cellulolytic isolates belonged to four phyla i.e. Fermicutes, Proteobacteria, Bacteroidetes 20

and Actinobacteria. Among these phyla, several genera such as Bacillus, Geobacillus, Paenibacillus, 21

Pseudomonas, Arthrobacter, Streptomyces, Pedobacter, Flavobacterium and Chryseobacterium, 22

Agromyces, Duganella, Stenotrophomonas, Microbacterium, Exiguobacterium and Laceyella have 23

shown cellulase activity. The fact high number of cellulase positive bacteria and their distribution to 24

diverse phylum in harsh environment of PCH region was encouraging and proof of concept of our 25

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sampling sites which were focused on decaying biological biomass. In high altitude region the 1

cellulose was accumulated in environment from various summer herbs and shrubs. In addition, activity 2

of migratory sheep and goat flocks of Gaddi community (a tribe of shepherds in Chamba district of 3

Himachal Pradesh, India) in PCH region might be the supplementary factor for the presence of 4

cellulosic biomass and cellulolytic bacterial community. In consistent with above notion, it has been 5

previously reasoned that ornithogenic activity of penguins is one of the reason for presence of cellulose 6

in Antarctica (Soares Jr et al. 2012). A few studies have reported the presence of cellulolytic bacteria 7

from harsh environment such as cold dry regions or high altitude (Soares Jr et al. 2012; Venkatahalam 8

et al. 2015). Predominance of bacterial genera belonging to Bacteroidetes, Firmicutes and 9

Actinobacteria from Antarctica and Caatinga region of Brazil (Soares Jr et al. 2012) and Firmicutes 10

and Actinobacteria from mountain ranges of Indian and Nepal Himalaya were reported for cellulase 11

activity (Venkatahalam et al. 2015). Collectively, over the past years, several genera such as 12

Micrococcus, Cellulomonas, Bacillus, Pseudomonas, Burkholderia, Citrobacter, Pandoraea, 13

Arthrobacter, Microbacterium, Geobacillus, Paenibacillus, Rhodothermus, have been reported for 14

cellulolytic potential (Hreggvidsson et al. 1996; Kuhad et al.2011; Sadhu et al. 2011; Behera et al. 15

2014). 16

Comparative analysis of reported genera in the present study with available information in 17

the literature has revealed the identification of new and more diverse set of bacterial species for 18

cellulolytic potential. Earlier, it was found that potential cellulase producing bacteria belonged to 19

genera Bacillus, Paenibacillus, Paracoccus, and Pseudomonas having HC values between 1.5 to 5.7 20

(Behera et al. 2014; Khianngam et al. 2014; Rathore. 2014). In the current study, bacterial isolates 21

with higher HC values (Table 2) for Flavobacterium sp. PCH74 (9), Flavobacterium sp. PCH81 22

(6.25), Geobacillus sp. PCH113 (6), Paenibacillus sp. PCH82 (5.5) were reported. Higher zone ratio in 23

CMC plates might be either due to compact cellulase system which can efficiently diffuse through the 24

medium or higher production/catalytic activity of this enzyme. These features of cellulase can be 25

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felicitous for tackling the obstacles in cellulose valorization bioprocesses. In addition to above, other 1

isolates that grow at a wide temperature and pH range could be equally important for harnessing 2

cellulase potential. 3

Since PCH region is virgin, unexplored and blessed with a unique habitat features, therefore, 4

possibility of finding novel bacterial isolates was anticipated. As expected, six putative novel bacteria 5

having 16S rDNA sequence similarity <98.6% and five other isolates in between 98.7% to 99% have 6

been discovered (Table 1). This suggested the uniqueness and unexplored nature of PCH region for 7

microbiological interventions. Similarly, four putative novel bacteria (16S rDNA sequence similarity 8

<97%) have been identified from other unique niches of Leh-Siachen region of western Himalaya. 9

Some unique taxa i.e. Ferribacterium, Rothia, Wautersiella, Erwinia, Mycetecola, Agromyces, 10

Kluyvera, and Enterobacter have also been reported from the Himalayan mountain ranges 11

(Venkatachalam et al. 2015). Thus, literature supports our hypothesis of high probability of finding 12

novel bacteria in high altitude region of western Himalaya. 13

Another important observation in present study is the isolation and identification of 14

thermophilic bacteria, despite the fact that soil and water samples were from cold environmental niches 15

of non-thermogenic origin (Table S1). The presence of high thermophilic bacterial diversity (14% of 16

total isolates) in the overall culturable bacterial community from such cold niches is actually 17

fascinating. Thermophiles, especially the members of genus Geobacillus have already been reported 18

from cold habitats such as glacial soil and ocean sediments (Marchant et al. 2002; Rahman et al. 2004; 19

Marchant et al. 2008; Muller et al. 2014). The tolerance of Geobacillus endospore in harsh conditions 20

like extreme temperatures and variable pH including quiescent and longevity is well documented for 21

the worldwide distribution (Zeigler 2014), and it might be the reason behind its high prevalence in 22

these regions. Besides Geobacillus, current study revealed few unique thermophiles belonging to 23

genera Bacillus, Streptomyces, Brevibacillus, Thermoactinomyces, Laceyella and Saccharococcus. 24

Previously such high number of thermophilic bacterial isolates has not been reported from colder 25

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niches. The newly identified thermophilic genera could be the important source for their applications 1

in ethanol production as comprehensively reviewed earlier (Arora et al. 2015). 2

Overall culturable bacterial community in PCH region revealed comparatively more 3

percentage of psychrotrophic (78%) and thermophilic (14%) bacteria to their mesophilic (8%) counter 4

parts. Earlier, distribution of psychrophilic /psychrotolerant (60%), mesophiles (36.5%) and 5

thermophilic bacteria (3.8%) had been reported in soil samples from western Himalaya. The 6

considerably higher amount of psychrotrophs and thermophiles in the present study compared to 7

earlier might be due to the unique topography of PCH region that has higher temperature fluctuation as 8

compared to Leh-Siachen region studied earlier (Gangwar et al. 2009). In addition, easily and 9

culturable group of bacteria such as Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes 10

were reported in consistent with earlier investigations (Gangwar et al. 2009; Gupta et al. 2015). 11

However, at phylum level unlike other investigations Firmicutes were reportedly abundant in the 12

present study (Fig. 2). Since, PCH region is very remote, rugged, virgin, and provide harsh 13

environmental conditions for survival of life; therefore, the abundance of spore forming Firmicutes 14

could be one the survival strategies to withstand and escape from the challenges posed by the 15

environments (Galperin 2013). The abundance of Acidobacteria, Proteobacteria and Actinobacteria 16

were also reported in the soil of Drass, a cold desert in Himalaya using 16S rRNA gene clone methods 17

(Gupta et al. 2015). The several studies conducted by Shivaji’s group (CCMB, Hyderabad, India) had 18

explored many Himalayan glaciers i.e. Roopkund, Pindari and Kafni for bacterial diversity by culture 19

independent method and revealed the dominance of Actinobacteria, Firmicutes and Proteobacteria 20

and also reported many other genera such as Acidobacteria, Bacteroidetes, Gemmatimonadetes, 21

Planctomycetes, Chloroflexi, Spirochaetes, Tenericutes and Verrucomicrobia (Stres et al. 2013; 22

Pradhan et al. 2010; Srinivas et al. 2011). Despite the abundance of Acidobacteria in various 23

Himalayan habitats, the current research did not find any member of this phylum; might be due to the 24

difficulties associated with their culturability (Kielak et al. 2016). Similarly, members of other 25

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bacterial phyla like Chloroflexi, Spirochaetae, Tenericutes and Verrucomicrobia were not obtained, 1

possibly due to their low abundance and difficulty in culturing. Permafrost in Chinese glaciers had rich 2

bacterial diversity and distributed into Actinobacteria, Firmicutes, Proteobacteria, and Cytophaga-3

Flavobacterium-Bacteroide (Hu et al. 2015). However, some recent studies on cold habitat across the 4

globe have found varied diversity (Lutz et al. 2015; Wang et al. 2016; Pudasaini et al. 2017). The 5

abundance of β-proteobacteria and Chloroflexi were observed in cryoconite samples of Karakoram 6

glacier and eastern Antarctica, respectively (Ambrosini et al. 2016; Ji et al. 2016). This has suggested 7

the geographical isolation and local environment is a major factor and key player influencing the 8

bacterial community ecology. It is well known that culture independent method is better and extensive 9

to study the microbial diversity, but culture-based study is more application oriented when exploring 10

for complex enzyme system such as cellulase for industrial purposes. 11

In conclusion, identification of diverse culturable bacteria with high cellulolytic potential can 12

be utilized for industrial applications. Further, studying their genomic insights in future will help us to 13

understand key adaptational features and to unlock their industrial potential. Evidently, high numbers 14

of thermophiles, especially, Geobacilli are reported and can be a rich source of thermostable enzymes. 15

Qualitative screening for cellulose degradation revealed a significant number of cellulolytic bacteria. 16

Some of them have shown very high zone ratio hitherto reported, thus, can be the exploited for the 17

valorization of cellulosic biomass to various industrial applications. Currently, enzymatic potential for 18

efficient cellulose degrader to develop a bioprocess for the commercial applications is being evaluated. 19

Author’s Contribution 20

SK and DS conceived the study, contributed reagents/materials. VT, VK, and DS conducted field 21

survey, the collection of samples, data analysis and wrote the manuscript. VT and DS designed and 22

performed the experiments. All authors read and approved the final manuscript. 23

Acknowledgements 24

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15

Authors acknowledge financial support from CSIR Network projects SIMPLE CeHAB (BSC0209) and 1

CSIR-IHBT in-house grant MLP071. Authors duly acknowledge the technical assistance provided by 2

Anil Chaudhary for 16S rDNA sequencing. This manuscript represents CSIR-IHBT communication 3

number 4162. 4

Conflict of Interest- None declared. 5

References 6

Albarracín, V.H., Pathak, G., Douki, T., Cadet, J. 2012. Extremophilic Acinetobacter Strains from 7

High-Altitude Lakes in Argentinean Puna: Remarkable UV-B Resistance and Efficient DNA 8

Damage Repair. Orig Life Evol. Biosph. 42: 201–221. 9

Ambrosini, R., Musitelli, F., Navarra, F., Tagliaferri, I. 2016. Diversity and assembling processes of 10

bacterial communities in cryoconite holes of a karakoram glacier. Microb. Ecol. 73: 827-837. 11

Arantes, V., Saddler, J.N. 2010. Access to cellulose limits the efficiency of enzymatic hydrolysis: the 12

role of a morphogenesis. Biotechnol. Biofuels. 3: 4. 13

Arora, R., Behera, S., Kumar, S. 2015. Bioprospecting thermophilic/thermotolerant microbes for 14

production of lignocellulosic ethanol: A future perspective. Renew. Sustainable Energy Rev. 51: 15

699-717. 16

Balan, V. 2014. Current challenges in commercially producing biofuels from lignocellulosic biomass. 17

ISRN Biotechnol. http://dx.doi.org/10.1155/2014/463074. 18

Behera, B.C., Parida, S., Dutta, S.K., Thatoi, H.N. 2014. Isolation and Identification of Cellulose 19

Degrading Bacteria from Mangrove Soil of Mahanadi River Delta and Their Cellulase Production 20

Ability. Am. J. Microbiol. Res. 2: 41-46. 21

Bhat, A., Ul-Hassan, S.R., Ahmad, N., Srivastava, N. 2013. Isolation of cold-active, acidic 22

endocellulase from Ladakh soil by functional metagenomics. Extremophiles. 17: 229–239. 23

Bischof, R.H., Ramoni, J., Seiboth, B. 2016. Cellulases and beyond: the first 70 years of the enzyme 24

producer Trichoderma reesei. Microb. Cell Fact. 15: 106. 25

Page 15 of 29



Draft

16

Carpenter, E.J., Lin, S., Capone, D.G. 2000. Bacterial activity in south pole snow. Appl. Environ. 1

Microbiol. 66: 4514–4517. 2

Cavicchioli, R., Charlton, T., Ertan, H., Mohd Omar, S. 2011. Biotechnological uses of enzymes from 3

psychrophiles. Microb. Biotechnol. 4: 449–460. 4

Cragg, S.M., Beckham, G.T., Bruce, N.C., Bugg, T.D.H. 2015. Lignocellulose degradation 5

mechanisms across the tree of life. Curr. Opin. Chem. Biol. 29: 108–119. 6

Elleuche, S., Schroder, C., Sahm, K., Antranikian, G. 2014. Extremozymes - biocatalysts with unique 7

properties from extremophilic microorganisms. Curr. Opin. Biotechnol. 29: 116–123. 8

Enzymes market size expected to reach $17.50 billion by 2024. 2016. 9

http://www.grandviewresearch.com/press-release/global-enzymes-market. Accessed on 22 July 10

2017. 11

Ferbiyanto, A., Rusmana, I., Raffiudin, R. 2015. Characterization and identification of cellulolytic 12

bacteria from gut of worker Macrotermes gilvus. HAYATI J. Biosci. 22: 197-200. 13

Galperin, M.Y. 2013. Genome Diversity of Spore-Forming Firmicutes. Microbiol. Spectr. 1: 2. 14

Gangwar, P., Alam, S.I., Bansod, S., Singh, L. 2009. Bacterial diversity of soil samples from the 15

western Himalayas, India. Can. J. Microbiol. 55: 564–577. 16

Gupta, P., Sangwan, N., Lal, R., Vakhlu, J. 2015. Bacterial diversity of Drass, cold desert in Western 17

Himalaya, and its comparison with Antarctic and Arctic. Arch. Microbiol. 197: 851–860. 18

Hasunuma, T., Okazaki, F., Okai, N., Hara, K.Y. 2013. A review of enzymes and microbes for 19

lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing 20

technology. Bioresour. Technol. 135: 513–522. 21

Hreggvidsson, G.O., Kaiste, E., Holst, O., Eggertsson, G., Palsdottir, A., Kristjansson, J.K. 1996. An 22

extremely thermostable cellulase from the thermophilic eubacterium Rhodothermus marinus. Appl. 23

Environ. Microbiol. 62: 3047-3049. 24

Hu, W., Zhang, Q., Tian, T., Cheng, G. 2015. The microbial diversity, distribution, and ecology of 25

permafrost in China: a review. Extremophiles. 19: 693–705. 26

Page 16 of 29



Draft

17

Jeoh, T., Cardona, M.J., Karuna, N., Mudinoor, A.R. 2017. Mechanistic kinetic models of enzymatic 1

cellulose hydrolysis-A Review. Biotechnol. Bioeng. 9999: 1-16. 2

Ji, M., Van Dorst, J., Bissett, A., Brown, M.V. 2016. Microbial diversity at Mitchell Peninsula, eastern 3

Antarctica: a potential biodiversity “hotspot.” Polar Biol. 39: 237–249. 4

Jordan, J., and Theegala, C. (2014) Probing the limitations for recycling cellulase enzymes 5

immobilized on iron oxide (Fe3O4) nanoparticles. Biomass Conv. Bioref. 4: 25–33. 6

Juturu, V., Wu, J.C. 2014. Microbial cellulases: Engineering, production and applications. Renew. 7

Sust. Energ Rev. 33: 188–203. 8

Kasana, R.C., Salwan, R., Dhar, H., Dutt, S. 2008. A rapid and easy method for the detection of 9

microbial cellulases on agar plates using gram’s iodine. Curr. Microbiol. 57: 503–507. 10

Khianngam, S., Pootaeng-on, Y., Techakriengkrai, T., Tanasupawat, S. 2014. Screening and 11

identification of cellulase producing bacteria isolated from oil palm meal. J. Appl. Pharm. Sci. 4: 12

90-96. 13

Kielak, A.M., Barreto, C.C., Kowalchuk, C.A., Veen, J.A. 2016. The ecology of Acidobacteria: 14

Moving beyond genes and genomes. Front. Microbiol. 7: 744. 15

Kim, M., Oh, H.S., Park, S., Chun, J. 2014. Towards a taxonomic coherence between average 16

nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. 17

Int. J. Syst. Evol. Microbiol. 64: 346–351. 18

Kuhad, R.C., Gupta, R., Singh, A. 2011. Microbial cellulases and their industrial applications. Enzyme 19

Res. http://dx.doi.org/10.4061/2011/280696. 20

Lutz, S., Anesio, A.M., Edwards, A., Benning, L.G. 2015. Microbial diversity on icelandic glaciers 21

and icecaps. Front. Microbiol. 6: 307. 22

Marchant, R., Banat, I.M., Rahman, T.J., Berzano, M. 2002. The frequency and characteristics of 23

highly thermophilic bacteria in cool soil environments. Environ. Microbiol. 4: 595–602. 24

Page 17 of 29



Draft

18

Marchant, R., Franzetti, A., Pavlostathis, S.G., Tas, D.O. 2008. Thermophilic bacteria in cool 1

temperate soils: are they metabolically active or continually added by global atmospheric transport? 2

Appl. Microbiol. Biotechnol. 78: 841–852. 3

Muller, A.L., Rezende, J., Hubert, C., Urup, K. 2014. Endospores of thermophilic bacteria as tracers of 4

microbial dispersal by ocean currents. ISME J 8: 1153–65. 5

Payne, C.M., Knott, B.C., Mayes, H.B., Hansson, H. 2015. Fungal cellulases. Chem. Rev. 115: 1308–6

1448. 7

Pradhan, S., Srinivas, T.N.R., Pindi, P.K., Kishore, H.H. 2010. Bacterial biodiversity from Roopkund 8

Glacier, Himalayan mountain ranges, India. Extremophiles. 14:377–395. 9

Pudasaini, S., Wilson, J., Ji, M., van Dorst, J. 2017. Microbial diversity of browning peninsula, eastern 10

Antarctica revealed using molecular and cultivation methods. Front. Microbiol. 8: 591. 11

Rahman, T.J., Marchant, R., Banat, I.M. 2004. Distribution and molecular investigation of highly 12

thermophilic bacteria associated with cool soil environments. Biochem. Soc. Trans. 32. 13

Rathore, P. 2014. Cellulase production by liquid state fermentation of Bacillus species isolated from 14

woody forest soil. IJRSB. 2: 8-13. 15

Sadhu, S., Saha, P., Mayilraj, S., Maiti, T.K. 2011. Lactose-enhanced cellulase production by 16

Microbacterium sp. isolated from fecal matter of Zebra (Equus zebra). Curr. Microbiol. 62: 1050–17

1055. 18

Sahay, H., Babu, B.K., Singh, S., Kaushik, R. 2013. Cold-active hydrolases producing bacteria from 19

two different sub-glacial Himalayan lakes. J. Basic Microbiol. 53: 703–714. 20

Salwan, R., Gulati, A., Kasana, R.C. 2010. Phylogenetic diversity of alkaline protease-producing 21

psychrotrophic bacteria from glacier and cold environments of Lahaul and Spiti, India. J. Basic 22

Microbiol. 50: 150-159. 23

Santiago, M., Ramírez-Sarmiento, C.A., Zamora, R.A., Parra, L.P. 2016. Discovery, molecular 24

mechanisms and industrial applications of cold-active enzymes. Front. Microbiol. 7: 1408. 25

Page 18 of 29



Draft

19

Sarmiento, F., Peralta, R., Blamey, J.M. 2015. Cold and hot extremozymes: Industrial relevance and 1

current trends. Front. Bioeng. Biotechnol. 3: 148. 2

Sharma, A., Tewari, R., Rana, S.S., Soni, R. 2016. Cellulases: Classification, Methods of 3

Determination and Industrial Applications. Appl. Biochem. Biotechnol. 179: 1346-1380. 4

Shivaji, S., Pratibha, M.S., Sailaja, B., Hara Kishore, K. 2011. Bacterial diversity of soil in the vicinity 5

of Pindari glacier, Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA 6

gene clones. Extremophiles, 15: 1–22. 7

Siddiqui, K.S., Cavicchioli, R. 2006. Cold-adapted enzymes. Annu. Rev. Biochem. 75: 403–433. 8

Singh, S., Moholkar, V.S., Goyal, A. 2013. Isolation, identification, and characterization of a 9

cellulolytic Bacillus amyloliquefaciens strain SS35 from Rhinoceros dung. ISRN Microbiol. 10

http://dx.doi.org/10.1155/2013/728134. 11

Soares Jr, F.L., Melo, I.S., Dias, A.C.F., Andreote, F.D. 2012. Cellulolytic bacteria from soils in harsh 12

environments. World J. Microbiol. Biotechnol. 28: 2195–2203. 13

Srinivas, T.N.R., Singh, S.M., Pradhan, S., Pratibha, M.S. 2011. Comparison of bacterial diversity in 14

proglacial soil from Kafni Glacier, Himalayan mountain ranges, India, with the bacterial diversity 15

of other glaciers in the world. Extremophiles. 15: 673–690. 16

Stres, B., Sul, W.J., Murovec, B., Tiedje, J.M. 2013. Recently deglaciated high-altitude soils of the 17

Himalaya: Diverse environments, heterogenous bacterial communities and long-range dust inputs 18

from the upper troposphere. PloS One. 8:e76440. 19

Valdivia, M., Galan, J.L., Laffarga, J., Ramos, J.L. 2016. Biofuels 2020: Biorefineries based on 20

lignocellulosic materials. Microb. Biotechnol. 9: 585–594. 21

Van den Burg, B. 2003. Extremophiles as a source for novel enzymes. Curr. Opin. Microbiol. 6: 213-22

218. 23

Venkatachalam, S., Gowdaman, V., Prabagaran, S.R. 2015. Culturable and culture-independent 24

bacterial diversity and the prevalence of cold-adapted enzymes from the Himalayan mountain 25

ranges of India and Nepal. Microb. Ecol. 69: 472–491. 26

Page 19 of 29



Draft

20

Wang, N.F., Zhang, T., Yang, X., Wang, S. 2016. Diversity and composition of bacterial community 1

in soils and lake sediments from an Arctic lake area. Front. Microbiol. 7: 1170. 2

Zeigler, D.R. 2014. The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a 3

mesophilic planet? Microbiology. 160: 1–11. 4

Tables 5

Table 1 Putative novel bacterial isolates from PCH region and their similarity (%) with type strains 6

analyzed in EzTaxon database. 7

Table 2 Potential bacterial isolates with high cellulose hydrolytic zone ratio and HC values for 8

cellulase activity. 9

Supplementary Tables 10

Table S1 Overview of bacteria isolated from PCH region along with their GenBank accession number 11

based on 16S rDNA sequences, growth conditions and qualitative cellulase activity (‘+’ means HC 12

value <1.0, ‘++’ means 1.0- 2.5 and ‘+++’ means >2.5). 13

Table S2 Qualitative screening of bacterial isolates for cellulase activity in terms of zone ratio and HC 14

values. 15

16

17

18

19

20

21

22

23

24

25

26

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Figure Legends 1

Fig. 1 Map showing the sampling sites in PCH region of Himachal Pradesh, India. 2

Fig. 2 Pie diagram representing the distribution of culturable bacterial phyla isolated from PCH region. 3

Fig. 3 Bacterial diversity among culturable bacteria of PCH region at genus level in their respective 4

four phyla. 5

Fig. 4 Phylogenetic tree of 16S rDNA sequences of bacterial isolates based on neighbor-joining 6

method. 7

Fig. 5 Comparative analysis of cellulase positive isolates in the respective phylum. 8

Fig. 6 Qualitative plate assay for cellulolytic activity showing zone of clearance in CMC agar plates. 9

Plate (A) showing negative control PCH8 (a), and PCH14(b), PCH17 (c), PCH52 (d) are 10

representative of potential isolates with moderate cellulase activity; and plate (B) showing isolates 11

PCH83 (e), PCH75 (f), PCH 74 (g) and PCH57 (h) with high cellulase activity. 12

13

14

15

16

17

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Table 1 Putative novel bacterial isolates from PCH region and their similarity (%) with type strains analyzed in

EzTaxon database.

S.

No.

Isolate

Code

Closest Match Similarity

(%)

1. PCH8 Paenibacillus amylolyticus NRRL NRS-

290(T)

96.90

2. PCH117 Saccharococcus thermophilus ATCC

43125 (T)

97.14

3. PCH22 Flavobacterium hercynium WB 4.2-33(T) 98.57

4. PCH34 Acinetobacter schindleri CIP 107287(T) 97.86

5. PCH35 Paenibacillus endophyticus PECAE04(T) 97.95

6. PCH74 Flavobacterium aquidurense WB-

1.1.56(T)

98.46

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Table 2 Potential bacterial isolates with high cellulose hydrolytic zone ratio and HC values for cellulase activity.

S.

No

Isolate

Code

Acc. No Closest match Ratio HC

values

1. PCH26 KY628844 Agromyces cerinus subsp.

nitratus ATCC 51763(T)

4.5 3.5

2. PCH60 KY628878 Paenibacillus xylanexedens

B22a(T)

4.5 3.5

3. PCH74 KY628892 Flavobacterium aquidurense

WB-1.1.56(T)

9.0 8.0

4. PCH75 KY628893 Chryseobacterium piperi

CTM(T)

5.0 4.0

5. PCH81 KY628899 Flavobacterium frigidimaris

KUC-1 (T)

6.25 5.25

6. PCH82 KY628900 Paenibacillus amylolyticus

NRRL NRS-290(T)

5.5 4.5

7. PCH83 KY628901 Exiguobacterium antarcticum

DSM 14480 (T)

4.33 3.33

8. PCH106 KY628924 Bacillus licheniformis ATCC

14580(T)

5.0 4.0

9. PCH113 KY628931 Geobacillus thermoleovorans

KCTC 3570 (T)

6.0 5.0

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Map showing the sampling sites in PCH region of Himachal Pradesh, India.

35x14mm (300 x 300 DPI)

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Pie diagram representing the distribution of culturable bacterial phyla isolated from PCH region.

43x38mm (300 x 300 DPI)

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Fig. 3 Bacterial diversity among culturable bacteria of PCH region at genus level in their respective four phyla

62x47mm (300 x 300 DPI)

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Phylogenetic tree of 16S rDNA sequences of bacterial isolates based on neighbor-joining method.

92x104mm (300 x 300 DPI)

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Comparative analysis of cellulase positive isolates in the respective phylum.

68x73mm (300 x 300 DPI)

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Qualitative plate assay for cellulolytic activity showing zone of clearance in CMC agar plates. Plate (A) showing negative control PCH8 (a), and PCH14(b), PCH17 (c), PCH52 (d) are representative of potential isolates with moderate cellulase activity; and plate (B) showing isolates PCH83 (e), PCH75 (f), PCH 74 (g)

and PCH57 (h) with high cellulase activity.

40x18mm (300 x 300 DPI)

Page 29 of 29



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