Diversity, cold active enzymes and adaptation strategies of bacteria inhabiting glacier cryoconite holes of High Arctic.

Extremophiles (2014) 18:229–242 DOI 10.1007/s00792-013-0609-6

ORIGINAL PAPER

Diversity, cold active enzymes and adaptation strategies of bacteria inhabiting glacier cryoconite holes of High Arctic Purnima Singh • Shiv M. Singh Prashant Dhakephalkar

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Received: 17 April 2013 / Accepted: 14 November 2013 / Published online: 18 December 2013 Ó Springer Japan 2013

Abstract Cryoconite holes have biogeochemical, ecological and biotechnological importance. This communication presents results on culturable psychrophilic bacterial diversity from cryoconite holes at Midre Loveńbreen (ML), Austre Brøggerbreen (AB), and Vestre Brøggerbreen (VB) glaciers. The culturable bacterial count ranged from 2.7 9 103 to 8.8 9 104 CFUs/g while the total bacterial numbers ranged from 5.07 9 105 to 1.50 9 106 cells at the three glaciers. A total of 35 morphologically distinct bacterial isolates were isolated. Based on 16S rRNA gene sequence data, the identified species belonged to eight genera namely Pseudomonas, Polaromonas, Micrococcus, Subtercola, Agreia, Leifsonia, Cryobacterium and Flavobacterium. The isolates varied in their growth temperature, NaCl tolerance, growth pH, enzyme activities, carbon utilization and antibiotic sensitivity tests. Fatty acid profiles indicate the predominance of branched fatty acids in the isolates. To the best of our knowledge, this is the first record of culturable bacterial communities and their Communicated by A. Oren.

Electronic supplementary material The online version of this article (doi:10.1007/s00792-013-0609-6) contains supplementary material, which is available to authorized users. P. Singh (&) Birla Institute of Technology and Science, Pilani-K.K. Birla Goa Campus, Zuarinagar, Goa 403726, India e-mail: [email protected] S. M. Singh National Centre for Antarctic and Ocean Research, Ministry of Earth Sciences, Vasco da Gama, Goa 403804, India P. Dhakephalkar Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India

characterization from glacier cryoconites from High Arctic. High amylase and protease activities expressed by Micrococcus sp. MLB-41 and amylase, protease and lipase activities expressed by Cryobacterium sp. MLB-32 provide a clue to the potential applications of these organisms. These cold-adapted enzymes may provide an opportunity for the prospect of biotechnology in Arctic. Keywords Bacteria Svalbard Glacier Diversity Biotechnological potentials

Introduction Cryoconite holes are organically rich, variously shaped, water filled depressions distributed over the glaciers. These holes are formed as the windblown low albedo microbial and mineral particles settle onto the glacier surface and melt down the surface ice to a depth of thermal equilibrium (McIntyre 1984; Paul and Clark 1989). Cryoconites can cover 0.1–10 % of the glacier surface (Anesio et al. 2009), can range in diameter from a few centimeters to more than a meter and can be up to 50 cm in depth (Gerdel and Drouet 1960; Fountain et al. 2008). Sa¨wstro¨m et al. (2002) reports the cryoconite holes in Svalbard to represent about 6 % of the glacier surface with an average of about 12 holes per m2. Typically, the cryoconite holes on the Svalbard glaciers are measured to be about 8–30 cm deep and 5–50 cm in diameter and to contain debris of about 0.1–1,000 g (Sa¨wstro¨m et al. 2002; Hodson et al. 2007). The cryoconite holes have two distinct zones—the pelagic and the benthic (Gerdel and Drouet 1960). While the pelagic zone consists of the meltwater, the benthic zone contains soft, small, spherical (0.1–3 mm in diameter), dark colored granular material, mainly of organic and

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inorganic nature (Gerdel and Drouet 1960; Takeuchi et al. 2001, 2010). The organic matter includes algae, cyanobacteria, bacteria, fungi, and rotifers (Xu et al. 2010), while the inorganic matter is a mixture of minerals and trace elements (Singh et al. 2012). Organisms within the cryoconites grow in the presence of nutrients leached from the mineral particles and form a unique and biologically active habitats within these ice entombed environments (Sa¨wstro¨m et al. 2002; Foreman et al. 2007). Cryoconite holes due to their importance in biogeochemistry and ecology form a focus area of research for biologists and glaciologists (Stibal et al. 2008; Hodson et al. 2010; Bagshaw et al. 2011; Stibal et al. 2012). In the Antarctic, generally, the cryoconites on glaciers remain frozen at the surface by about 30 cm thick ice, insulating the contents from the influence of atmosphere and meltwater for as long as 10 years (Tranter et al. 2004; Hodson et al. 2008). Few of the holes get exposed to the atmosphere only during a portion of the austral summer (Wharton et al. 1985; Mueller et al. 2001). In contrast, the Arctic and Alpine cryoconite holes generally remain open to the atmosphere during each summer, allowing the exchange of gases, nutrients and biological propagules (Hodson et al. 2007). Microscopy-based approaches toward cryoconite studies in the Arctic began with the study of brown polycellular algae by Leslie (1879). Later, cyanobacteria (Stibal et al. 2006; Kastovska et al. 2005), viruses and virus-like particles (Anesio et al. 2007; Sa¨wstro¨m et al. 2002) and diatoms (Yallop and Anesio 2010) were studied from Arctic glacier cryoconites. Pursuing the non-culturable approach, Edwards et al. (2011, 2013) examined the distributions of bacterial communities in cryoconite holes from the Austre Brøggerbreen, Midre Loveńbreen and Vestre Brøggerbreen glaciers in Svalbard and observed that significantly different communities occurred in the cryoconite holes of cold valley glacier, Austre Brøggerbreen as compared to the two polythermal glaciers. Recently, yeast and filamentous fungi of cryoconite holes of Arctic have been characterized (Singh and Singh 2011). Culturable bacterial diversity of the cryoconite holes and their biotechnological potentials remain undescribed so far from Arctic. With the review of literature, it is clear that cryoconite habitats of Arctic are a focus area of research for culturable bacterial diversity and need investigation. Antarctic cryoconites were studied for the presence of bacteria, cyanobacteria, protozoa and rotifers (Porazinska et al. 2004). The culture-based techniques have been used for cryoconite holes to study bacterial diversity from Antarctic (Christner et al. 2003), and Alpine glacier (Lee et al. 2011). Bipolar comparison of cryoconites for algal and cyanobacterial communities was done by Mueller et al. (2001) and Mueller and Pollard (2004). Cameron et al.

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(2011) explored the structure and bacterial, eukaryotic and archaeal diversity of cryoconite communities from multiple Arctic and Antarctic locations using T-RFLP-based approach concluding that the polar cryoconite holes harbor geographically distinct highly diverse communities, many species of which may be hitherto unknown. The aims of current study were, first, to investigate whether cryoconite hole microbial communities exhibited spatial variability within the three glaciers of High Arctic. Secondly, to characterize the physiological features of dominant bacterial strains and screen them for their biotechnological potentials. This is the first study on antibiotic resistance from the Arctic cryoconite isolates. Together, these studies are aimed to improve our understanding of the diversity and possible origins and functions of microbial communities in cryoconite holes. In the biotechnological perspective, they can be looked at as potential sources of cold-adapted enzymes and other metabolites.

Materials and methods Sampling site Two polythermal valley glaciers, Midre Loveńbreen (ML) and Vestre Brøggerbreen (VB), and one cold valley glacier, Austre Brøggerbreen (AB) (Hagen and Satrang 1991) located in the Kongsfjord region of northwest Spitsbergen (78°530 N, 12°040 E) were investigated (Figs. 1, 2a). ML and AB have been studied intensively for mass balance since late 1960s (Hagen et al. 2003). During Arctic summer months, sediment samples were collected from five cryoconite holes (Fig. 2b, c) on each of these three glaciers. The water temperature in the cryoconite holes was between 0.2 and 1.9 °C and pH 7.1–8.6 (measured using Thermo Orion 4 Star, USA). The organic matter in the holes is loose, rounded and brownish black in color. Cryoconite samples were harvested by aspirating spherical dark colored granular material (debris) with a sterile 50 ml syringe into sterile tubes, placed in sterile ampules (Himedia) and stored at low temperature (-20 °C) until analyses. The samples were processed within 48 h of collection. Total bacterial counts Total bacterial number was estimated following the procedure of Kuwae and Hosokawa (1999). One gram organic matter was aseptically added to 5 ml of filter-sterilized saline and vortexed thoroughly to dislodge the microbial cells from the sediment grains. One milliliter of the suspension was added to 20 ll DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride; Himedia) solution (0.25 % w/v in sterile


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Fig. 1 An outline map of study ˚ lesund on the coast sites in Ny-A of Spitsbergen, and cryoconite sampling locations (star circle) over the glaciers (Midre Loveńbreen, Vestre Brøggerbreen and Austre Brøggerbreen)

distilled water) taken in a sterile vial. After incubating in the dark for 30 min, the sample was filtered on to a 0.2 lm Nuclepore polycarbonate track-etched membrane filter. The unbound DAPI was washed off by rinsing the filter with a few drops of saline. The membrane filter was observed under epifluorescent microscope (BX-51, Olympus, Japan), using non-fluorescent immersion oil. Bacterial cells appearing as bright blue spots against a dark background were enumerated and the cell number per gram soil was calculated. Isolation and culturing of bacteria One gram of sediment from cryoconite holes was suspended in 9 mL sterile saline and diluted serially (decimal dilutions 10-1, 10-2 and 10-3). Enumeration of culturable

bacteria was done using the spread plate method (0.1 ml) on nutrient agar (NA), 1/10 nutrient agar (1/10 NA), marine broth (MB), 1/10 marine broth (1/10 MB), Antarctic bacterial medium (ABM) and 1/10 Antarctic bacterial medium (1/10 ABM) and incubated at two different temperatures 4 and 15 °C for 14–30 days. ABM contains (per liter) 5 g peptone, 2 g yeast extract and 15 g agar, pH 7.0 (Reddy et al. 2000). Colony forming units (CFUs) appearing after incubation were counted and CFU number per gram soil was calculated. Isolates for the study were picked both based on unique morphotypes and also randomly from each plate. The pure isolates obtained were stored in glycerol stock at -70 °C and the most promising isolates MLB-32 (JCM 19504) and MLB-41 (JCM 19508) deposited at RIKEN-BRC JCM, Japan.

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Fig. 2 a Landscape of Svalbard Arctic showing the valley glaciers. b ‘Cryoconite hole’ exposed after the melt of soft ice during Arctic summer. c Manually opened a cryoconite hole before sampling. d Bacterial colony grown on culture media at 4 °C for 30 days

Phenotypic characterization of the bacterial strains Colony characteristics were observed with the help of a magnifying lens. Pure cultures obtained after 2–3 subcultures were subjected to physiological, carbon utilization, enzyme production and antibiotic sensitivity tests. Carbon utilization test was carried out using High Carbo kits (Himedia). Growth at different temperatures, pH and salt concentrations was carried out on ABM plates. The isolates were screened for enzymatic activities such as amylase, cellulase, lipase, protease and urease after incubation for 2 weeks at 4, 10 and 20 °C on appropriate media. Catalase was also tested at 4 °C. Amylase activity was determined on plates containing (per liter) 5 g soluble starch, 1 g peptone, 1 g yeast extract and 18 g agar. After 2 weeks of incubation, starch hydrolysis was determined with iodine staining (Smibert and Krieg 1994). Cellulase activity was screened on plates containing (per liter) 5 g carboxymethyl cellulose, 1 g peptone, 1 g yeast extract and 18 g agar. After 2 weeks of incubation, the plates were treated with Congo red and NaOH as described by Teather and Wood (1982). Protease activity was determined on plates containing (per half liter) 12 g casein, 6 g peptone, 6 g yeast extract and 9 g agar. Clearing zones around the colonies indicated protease activity. Lipase was determined on Tween 80 ? CaCl2 plates containing (per liter) 10 ml Tween 80, 1 g peptone, 1 g yeast extract, 1 g CaCl2 and 18 g agar. Formation of opaque halo around colonies indicated lipase activity (Smibert and Krieg 1994). Urease activity was tested on YNBG (6.7 g/l Yeast Nitrogen Base, 20 g/l glucose), containing 1 g/l

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urea (pH 5.5). Change in color from orange to pink indicated positive activity. Catalase activity was tested using a drop of H2O2. Formation of bubbles indicated positive activity. Molecular characterization DNA extraction Representative isolates were selected for molecular characterization (Table 1). The cultures initially isolated on different bacteriological media were all grown on Nutrient Agar plate for 10–15 days at 4 °C. They were subjected to total DNA extraction using the Bacterial InstaGene DNA Extraction Kit (BIO-RAD Laboratories) following manufacturer’s instructions. Polymerase chain reaction (PCR) and sequencing Extracted DNA was amplified by PCR method using KODplus DNA polymerase (TOYOBO Co, Ltd, Osaka, Japan). The 16S rRNA gene was amplified using the primers: 9F (GAGTTTGATCCTGGCTCAG) and 1541R (AAGGAGG TGATCCAGCC). Sequences were obtained with ABI prism 3100 Sequencer (Applied Biosystems) using ABI standard protocol. Sequence alignment and phylogenetic analysis Sequence alignment of 16S rRNA gene of each isolate was performed using Clustal W option of the software

Flavobacterium sp. NA-d

Leifsonia sp. NA-1 Leifsonia sp. MB-3 Cryobacterium sp. MB-10 Leifsonia sp. MB-12 Leifsonia sp. NA–k

NA-da

NA-1a MB-3a MB-10a MB-12a NA–ka ND ND ND ND ND

ND

JF790967 JX205209 JX205212 JX205210 JX205214 JX205211 JX205208 JF790969 JX205212 JX205195 JF790970 JX205207 JF790971 JF790968 JX205198 JX205199 JX205200 JX205203 JX205204 JX205205 JX205206 JX205202 JF790972 JX205196 JX205197 JX205201 JF790966 JF790973 ND

Sequence deposition no.

Sequence not used in phylogenetic tree ([1000 bp)

Pseudomonas sp. MB-g Pseudomonas sp. MLB-42 Pseudomonas sp. MLB-46 Pseudomonas sp. MLB-43 Polaromonas sp. MLB-45 Polaromonas sp. MLB-44 Micrococcus sp. MLB-41 Micrococcus sp. MB-18 Subtercola sp. MLB-47 Agreia sp. MLB-28 Agreia sp. MB-6 Leifsonia sp. MLB 40 Leifsonia sp. NA-8 Cryobacterium sp. MB-l Cryobacterium sp. MLB-31 Cryobacterium sp. MLB-32 Cryobacterium sp. MLB-33 Cryobacterium sp. MLB-36 Cryobacterium sp. MLB-37 Cryobacterium sp. MLB-38 Cryobacterium sp. MLB-39 Cryobacterium sp. MLB-35 Cryobacterium sp. MB-5 Cryobacterium sp. MLB-29 Cryobacterium sp. MLB-30 Cryobacterium sp. MLB-34 Cryobacterium sp. NA-9 Leifsonia sp. NA-b Cryobacterium sp.NA-i

MB-g MLB 42 MLB 46 MLB 43 MLB 45 MLB 44a MLB 41 MB-18 MLB 47 MLB-28 MB-6 MLB 40 NA-8 MB-l MLB 31 MLB 32 MLB 33 MLB 36 MLB 37a MLB 38 MLB 39 MLB 35 MB-5 MLB 29 MLB 30 MLB 34 NA-9a NA-ba NA-ia

a

Identification

Isolate Code

60.53 63.33 63.89 62.80 63.65

37.11

61.33 59.42 54.66 50.89 62.02 62.72 ND ND 58.88 ND ND 60.41 ND ND 59.88 59.90 57.96 63.44 63.12 ND 62.18 63.73 60.12 55.72 59.96 60.36 63.22 60.40 63.09

Mol G?C %

Table 1 Identification of bacterial isolates by sequences similarity (%)

650 500 550 450 500

700

1433 1482 1377 1474 1383 981 1415 1365 1444 1436 1421 1389 1252 1414 1470 1473 1479 1418 686 1424 1424 1456 1289 1419 1383 1298 799 687 600

Total sequence length after alignment

ND ND ND ND ND

ND

45 46 37 32 31 10 3 12 32 65 75 22 53 64 64 52 63 26 3 64 23 4 22 26 27 6 16 5 ND

No. of base changes

Pseudomonas ficuserectae JCM2400T (AB021378) by 96.9 % Pseudomonas ficuserectae JCM2400T (AB021378) by 96.9 % Pseudomonas ficuserectae JCM2400T (AB021378) by 97.3 % Pseudomonas ficuserectae JCM2400T (AB021378) by 97.8 % Polaromonas naphthalenivorans CJ2T (AY166684) by 97.8 % Polaromonas naphthalenivorans CJ2T (AY166684) by 99.0 % Micrococcus endophyticus YIM56238T (EU005372) by 99.8 % Micrococcus endophyticus YIM56238T (EU005372) by 99.1 % Subtercola frigoramans DSM1305T(AM410673) by 97.8 % Agreia pratensis DSM 14226T (AJ310412) by 95.5 % Agreia pratensis DSM 14226T (AJ310412) by 94.7 % Leifsonia antarctica SPC 20T (AM931710) by 98.4 % Leifsonia antarctica SPC 20T (AM931710) by 95.8 % Cryobacterium psychrophilum DSM4854T(AM410676) by 95.5 % Cryobacterium psychrophilum DSM4854T(AM410676) by 95.6 % Cryobacterium psychrophilum DSM4854T(AM410676) by 96.5 % Cryobacterium psychrophilum DSM4854T(AM410676) by 95.7 % Cryobacterium psychrotolerans hp36 (JN637331) by 98.2 % Cryobacterium psychrotolerans hp36 (JN637331) by 99.6 % Cryobacterium psychrotolerans hp36 (JN637331) by 95.5 % Cryobacterium psychrotolerans hp36 (JN637331) by 98.4 % Cryobacterium sp.DR9 (FJ464984) by 99.7 % Cryobacterium sp.DR9 (FJ464984) by 98.3 % Cryobacterium psychrotolerans hp36 (JN637331) by 98.2 % Cryobacterium psychrotolerans hp36 (JN637331) by 98.0 % Cryobacterium psychrotolerans hp36 (JN637331) by 99.5 % Cryobacterium sp. DR9 16S ribosomal RNA gene (FJ464984) by 98.0 % Leifsonia sp. 4-69 16S ribosomal RNA gene (GU213322) by 99.3 % Cryobacterium sp. Lc30-4 16S ribosomal RNA gene, partial sequence (GU733465) by 97 % Flavobacterium degerlachei strain LMG 21915 16S ribosomal RNA (AJ557886) by 97 % Leifsonia sp. 4-16 16S ribosomal RNA gene (GU213305) by 98 % Leifsonia sp. 4-69 16S ribosomal RNA gene (GU213322) by 94 % Cryobacterium sp. 4-57 16S ribosomal RNA gene, partial sequence by 98 % Leifsonia sp. 3030 16S ribosomal RNA gene by 96 % Leifsonia sp.

16S rRNA gene sequences similarity (%)

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Molecular Evolutionary Genetics Analysis (MEGA) software v4.0. (Tamura et al. 2007). The sequences of 28 isolates were deposited in the DNA data bank (NCBI) and were subjected to a NCBI BLAST search. The 16S rRNA gene sequences were aligned using Clustal W together with the homologous sequences (retrieved from Genbank) of closely related species of bacteria. To calculate the sequence similarity for 16S rRNA gene, the matrix was analyzed with the neighbor-joining method (Saitou and Nei 1987) using the Tamura–Nei model (Tamura and Nei 1993) and Maximum Parsimony method (Tamura et al. 2011). The bootstrap consensus tree (Felsenstein 1985) was inferred from 1000 replicates to represent the evolutionary history of the taxa analyzed.


Results Characteristics of the glacier cryoconite samples The pH in the cryoconite holes varied depending upon the open, closed and interconnected channels where water is regularly recycled by melting of surface waters. At ML and VB, the pH was between 7.1 and 7.9, while at AB it was between 7.6 and 8.6. Water temperature also varied, with ML and VB having a water temperature of 1.0–1.9 °C and AB having a water temperature of 0.8–1.2 °C. At all the three glaciers, organic carbon ranged from 1.07 to 2.5 %, nitrate content from 44 to 50 lg/g, mineralizable nitrogen from 0.27 to 0.32 mg/g and total phosphorus from 0.35 to 0.44 mg/g. Bacterial count

FAME analysis For fatty acid methyl ester analysis, the isolates were all grown on nutrient broth for 7–10 days at 4 °C. Whole-cell fatty acids were extracted from 100 mg of freeze dried culture using acid methanolysis method and analyzed by GLC equipped with flame ionization detector (model GC353, GL Science) at an initial temperature of 140 °C for 15 min and then increased to 240 °C at an increment of 4 °C min-1 (Yumoto et al. 2001). Fatty acids were identified by comparison with FAME mix standards (Supelco 37 component FAME mix).

The culturable bacterial count (colony forming unit, CFU) in the cryoconite holes was tested on various bacteriological media (NA, 1/10 NA, MB, 1/10 MB, ABM and 1/10 ABM). It ranged from 2.7 9 103 to 3.02 9 104 (mean 1.13 9 104 ± 0.84 9 104) per gram at ML, 2.3 9 104 to 8.8 9 104 (mean 4.97 9 104 ± 3.40 9 104) at AB and 3.00 9 104 to 3.1 9 104 (mean 3.02 9 104 ± 0.04 9 104) at VB glaciers. The total count (TC) of bacteria through DAPI showed different trends at ML [5.07 9 105 to 11.9 9 105 (mean 8.16 9 105 ± 3.07 9 105)], AB [1.24 9 106 to 1.50 9 106 (mean 1.34 9 106 ± 0.14 9 106)] and VB [1.17 9 106 to 1.33 9 106 (mean 1.24 9 106 ± 0.08 9 106)] glaciers.

Antibiotic susceptibility test Characteristics of the isolated strains Antibiotic susceptibility of the isolates was tested using the impregnated disc method (Bauer et al. 1966) with slight modification. All cultures were grown in nutrient broth at 15 °C up to a turbidity of 0.08–0.13 optical density at 620 nm. 100 ll of the bacterial suspensions was spread onto the surface of Mueller–Hinton Agar medium using sterile spreaders. Antibiotic discs were placed over bacterial lawn and the plates were incubated at 15 °C for 3–4 days. After the incubation period, plates were observed for formation of zone of inhibition, if any. The zone formed was measured in millimeters. Statistical analyses Simpson’s (1-D) and Shannon’s (H) diversity and evenness (eH/S) indices for each of the glaciers were calculated using the PAST software ver. 2.01 (Hammer et al. 2001) to compare the bacterial diversity in the cryoconite samples of the 3 glaciers.

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Of the numerous CFUs appearing on the culture media plates, 22 distinct representative isolates were purified for further studies (Supplementary Table 1). Of these 6 isolates were from ML, 4 from AB and 12 from VB. The culture colonies were of different colors—white, cream, yellow, orange and gray (Fig. 2d). The colonies were characterized by entire margin; opaque and smooth texture; and convex, pulvinate to raised elevation. The shapes of bacterial cells were cocci and short to long rods. The rodshaped bacteria varied in size from 0.15 to 0.5 lm 9 0.07 to 0.17 lm, while the cocci forms varied from 0.22 to 0.25 lm in diameter. Most of isolates showed growth temperature ranging from 1 to 22 °C, except one isolate at 30 °C. Optimum temperature for growth was 15 °C. The representative strains were, therefore, obligately psychrophilic in nature. The isolates showed salt tolerance at 2 and 5 % with no growth at 7 % salt concentration. Testing the growth of the isolates at various pH values showed that pH 6.0–7.0 is optimum for the growth of the cultures.


Antibiotic resistance patterns of the isolated strains When the isolates were subjected to antibiotic screening, it was observed that all the isolates were sensitive to antibiotics such as Amikacin AK30, Cefotaxime, Doxycyline hydrochloride, Kanamycin, Levofloxacin, Lomefloxacin, Piperacillin, Polymyxin-B and Tetracycline (Supplementary Table 2). The sensitivity to other antibiotics tested varied from isolate to isolate. Methicillin was the least potent of antibiotics tested, sensitive to only two isolates (MLB-40 and NA-9). Of the various isolates, screened NA-9 was the most sensitive having sensitivity toward all the antibiotics screened, while MLB-31 was the most resistant showing resistance to 24 of the 45 antibiotics screened. Other isolates showed varied resistance profile toward the antibiotics. Carbon utilization ability of the isolated strains Of the various carbon sources tested, most isolates (19) assimilated mannose sugar, 18 could hydrolyze esculin while 17 each could utilize rhamnose and xylose sugars (Supplementary Table 3). None of the isolates were capable of utilizing compounds such as sodium gluconate, erythritol, a-methyl-D-glucoside, melezitose, a-methyl-Dmannoside or sorbose. Amongst the isolates tested, MLB-42 has the ability to utilize as much as 27 of the 35 carbon sources tested, while MLB-32 utilized only 3 carbon sources (esculin, citrate and malonate) of those tested. Extracellular enzymatic activities Screening of the cultures for enzyme production indicated that all the 21 cultures were catalase positive, 16 of the isolates were positive for amylase, 13 for cellulase, 12 for lipase, 11 for urease and 6 for protease (Supplementary Table 4). Temperature played an important role in enzyme production. In case of amylase and urease, enzyme production increased with increase in temperature from 4 to 20 °C while in case of cellulase, enzyme production decreased with increase in temperature. For the rest of the enzymes (lipase and protease), the activity varied. MLB-32 was the most promising culture as it was capable of producing all the enzymes tested while 6 other cultures (MLB 45, MLB 41, NA-b, MB-g, MLB 33, MLB 46) were able to produce 5 of the different enzymes tested. Five cultures (MLB 42, MLB 40, MLB 47, MLB 29, MLB 33) produced only 2 of the enzymes tested. Fatty acid profiles Nine isolates (4 belonging to genus Cryobacterium, 2 each to Micrococcus and Agreia and 1 to genus Leifsonia) were

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subjected to fatty acid analysis. The fatty acids analyzed were grouped as saturated (C12:0, C14:0, C16:0, C18:0), unsaturated (C17:1x9c, C18:1x9c, C18:3x6,9,12c) and branched (iso C11:0 3OH, anteiso-C13:0, iso C14:0, iso C15:0, anteiso C15:0, iso C15:1 G, anteiso C15:10 A, iso C16:0, iso C17:0, anteiso C17:0, iso C17:1x5c, iso C18:1 H). It was observed that in all the isolates the concentration of branched fatty acids was much higher than the saturated or unsaturated fatty acids. The most dominant fatty acids recorded in the isolates are anteisoC15:0, anteiso-C17:0 iso-C15:0, iso-C16:0 and anteiso-C15:1 A. Phylogenetic analyses/taxonomic analysis The total sequence lengths after alignment, % sequence similarities, number of positions with base changes and the NCBI sequence deposition numbers are given in Table 1. In the pairwise alignment performed using EMBOSS Matcher–Pairwise Sequence Alignment tool (http://www.ebi.ac.uk/Tools/psa/emboss_matcher/nucleotide. html), the sequence analysis of the 16S rRNA gene domain of isolate Pseudomonas sp. MB-g (JF790967), Pseudomonas sp. MLB-42 (JX205209), Pseudomonas sp. MLB-46 (JX205212) and Pseudomonas sp. MLB-43 (JX205210) indicated their closest relationship to species of Pseudomonas oryzihabitans IAM1568T (AM262973) and Pseudomonas ficuserectae JCM2400T (AB021378). Polaromonas sp. MLB-45 (JX205214) indicated their closest relationship to species of Polaromonas naphthalenivorans CJ2T (AY166684) and Polaromonas jejuensis NBRC106434T (AB682445). Micrococcus sp. MLB-41 (JX205208) and Micrococcus sp. MB-18 (JF790969) resembled Micrococcus endophyticus YIM56238T (EU005372), Micrococcus yunnanensis YIM65004T (FJ214355), Micrococcus luteus DSM20030T (AJ536198) and Micrococcus lylae DSM20315T (X80750). Subtercola sp. MLB-47 (JX205212) resembled Subtercola frigoramans DSM1305T (AM410673) and Agreia pratensis DSM 14226T (AJ310412). Sequence analysis of Agreia sp. MLB-28 (JX205195) and Agreia sp. MB-6 (JF790970) showed closest relationship with Agreia pratensis DSM 14226T (AJ310412) and Subtercola frigoramans DSM1305T (AM410673). Sequence analysis of Leifsonia sp. MLB-40 (JX205207) and Leifsonia sp. NA-8 (JF790971) resembled Leifsonia antarctica SPC -20T (AM931710) and Leifsonia kafniensis KFC-22T (AM889135). Sequence analysis of isolates Cryobacterium sp. MLB-32 (JX205199), Cryobacterium sp. MLB-31 (JX205198), Cryobacterium sp. MLB-33 (JX205200), Cryobacterium sp. MB-l (JF790968) indicated their closest relationship to the species of Cryobacterium psychrophilum DSM4854T (AM410676). Sequence analysis of isolates Cryobacterium sp. MLB-38 (JX205205), Cryobacterium sp. MLB-39 (JX205206) and

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Fig. 3 Phylogenetic tree shows the cryoconite isolates with closely related reference species based on the 16S rRNA gene sequences. The accession numbers of strains are shown in parentheses. Tree was constructed with neighborjoined method using the Jukes– Cantor distance estimation. The significance of each branch is indicated by a bootstrap value. The scale bar is estimated substitutions per nucleotide position

Cryobacterium sp. MLB-36 (JX205203) resembled Cryobacterium psychrotolerans hp36 (JN637331), and Cryobacterium sp.DR9 (FJ464984). Cryobacterium sp. MLB-35 (JX205202) resembled Cryobacterium sp.DR9 (FJ464984) and Cryobacterium psychrotolerans hp36 (JN637331). Cryobacterium sp. MLB-29 (JX205196), Cryobacterium sp. MLB-

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30 (JX205197), Cryobacterium sp. MLB-34 (JX205201) and Cryobacterium sp. MB-5 (JF790972) resembled with Cryobacterium sp.DR9 (FJ464984) and Cryobacterium psychrotolerans hp36 (JN637331). The results have been elaborated using the neighbor-joining phylogenetic tree (Fig. 3) along with bootstrap support values for 1000 replications.


Distribution of the isolates in the cryoconites of the three glaciers The bacterial isolates from the cryoconites of three glaciers belonged to eight different genera namely, Pseudomonas, Polaromonas, Micrococcus, Subtercola, Agreia, Leifsonia, Cryobacterium, and Flavobacterium (Table 1). Number of taxa at each glacier varied from 3 to 8, while number of isolates varied from 5 to 17. Evenness index ranged from 0.912 (VB) to 0.976 (ML); Simpson’s diversity index (1-D) from 0.64 (AB) to 0.85 (VB); and Shannon’s diversity index (H) from 1.05 (AB) to 1.99 (VB). Pseudomonas ficuserectae (GC 50.89–59.42 %), Agreia pratensis, Leifsonia antarctica (GC 60.41 %) and Cryobacterium psychrotolerans (GC 59.96 %) were isolated from ML, Cryobacterium psychrotolerans (GC 55.72–60.36 %) and Subtercola frigoramans (GC 58.88 %) were isolated from AB; and Pseudomonas ficuserectae (GC 54.66–61.33 %), Polaromonas naphthalenivorans (GC 62.02–62.72 %), Micrococcus endophyticus, Cryobacterium psychrophilum (GC 57.96–59.9 %), C. psychrotolerans (GC 62.18–63.44 %) and Flavobacterium degerlachei (GC 37.11 %) were isolated from VB. The most predominant species was Cryobacterium psychrotolerans followed by C. psychrophilum (Table 1). Cryobacterium psychrotolerans was common to all glacier cryoconites. Isolates affiliated to genus Pseudomonas were the next most abundant. Pseudomonas ficuserectae exhibited discontinuous distribution along the different glacier cryoconites and were not obtained on the AB glacier cryoconite. Isolates affiliated to taxon Leifsonia antarctica which were all present in ML glacier and absent in AB and VB glaciers. Genera Polaromonas, Micrococcus, Subtercola, Agreia, Flavobacterium were represented the least. Polaromonas naphthalenivorans and Micrococcus endophyticus were present in only VB glacier; Subtercola frigoramans was present in only AB glacier. Agreia pratensis was present in only ML glacier and Flavobacterium degerlachei was present in only VB glacier. Glacier VB showed maximum diversity representing 8 different species, while AB glacier showed minimal diversity representing only 3 species. The results indicate that distribution of taxa did not exhibit any specific pattern of distribution along the different glacier cryoconites. These results, however, were strictly based on culturable approach and abundance of non-culturable species cannot be denied.

Discussion In the present study, the pH of the cryoconite holes was reported between 7.1 and 8.6. Cryoconite hole pH is unique and independent from one another, varying from glacier to

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glacier and sometimes even from hole to hole depending on the distance by which they are separated. The mineral contents and active photosynthesis influence the pH values of cryoconite holes (Tranter et al. 2004). Kastovska et al. (2005) have recorded the pH of cryoconite holes on Svalbard glaciers to be about 4.7, while the cryoconite holes on the Canada glacier (Antarctica) and the White glacier (Arctic) have been reported to be 5.9–9.6 and 3.4–4.5, respectively (Mueller and Pollard 2004). The nitrate content in the glaciers under study was between 44 and 50 lg/g while at White glacier, Canada (Mueller and Pollard 2004) it was quite low (\10–33 lg per liter). Nitrogen fixation in the cryoconite holes is done by the cyanobacterial communities present there (Vincent and Howard-Williams 1994) while the phosphorus inputs mainly come from atmospheric, hydrologic and mineral resources (Mueller et al. 2001). The organic carbon content in the study area was low (1.07–2.5 %) similar to that reported by Kastovska et al. (2005) and by Takeuchi et al. (2001) from other cryoconite samples, confirming the oligotrophic nature of this habitat. Total bacterial count in the cryoconite sediments of the three glaciers ranged from 5.07 9 105 to 1.50 9 106 per gram. These values were much lower than those recorded in a earlier study by Kastovska et al. (2005), in which TC was 7525.3 9 1011 per gram at ML, 538.0 9 1011 to 1717.1 9 1011 cells per gram at AB and 4342.8 9 1011 to 7192.7 9 1011 cells per gram at VB glaciers. This indicates that age, size and chemistry of the cryoconite holes are key factors that determine the colonization by bacteria. The bacterial CFUs that emerged on the petriplates ranged from 2.7 9 103 to 8.8 9 104 per gram at the three different glaciers studied (ML, AB and VB). No previous literature on bacterial CFU data of cryoconite sediments is available for comparison. A study on cryoconite meltwater at Antarctic glaciers records a maximum number of 8.6 9 104 CFU’s per ml (Christner et al. 2003). Non-culturable approach in diversity studies documents larger assortment of microbes. Culture-based approach, as applied in this study, however, in some respects, has benefits over culture-independent approaches. It is not possible to determine and compare the physiological characteristics of individual species within the samples using molecular approach. Further, the characterization of culturable microorganisms can provide information on the ecological roles of at least some members of the community, and thus may supplement to our knowledge of understanding community structure derived from direct molecular approaches (Jiang et al. 2006). In addition, a culture-based approach is required to assemble a collection of microorganisms on which to conduct biochemical, genetic and physiological experiments, and within which to observe inter- and intraspecies interactions (Jiang et al. 2006). To maximize the

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recovery of isolates, a number of bacteriological media of different strengths were used in the present study. Characteristics of the isolated strains The isolates were capable of growing at 1–22 °C, except for one which could grow even at 30 °C. The optimum temperature of growth for all, however, was 15 °C. The present observation showed that these bacterial isolates are strictly psychrophilic in nature. During a similar study with bacterial isolates from Antarctic cryoconite samples, Christner et al. (2003) observed that the isolates grew profusely at 4 and 15 °C as compared to 22 °C. On the contrary, cryoconite studies from the alpine glaciers by Lee et al. (2011) indicated that the bacterial isolates were predominantly psychrotolerant with a frequency peak at 30 °C. Antibiotic resistance patterns of bacteria In the present study, we observed that the sensitivity of isolates toward antibiotics varied. In comparison with the antibiotic testing studies carried in the tropical and temperate regions in the world, it can be said that the sensitivity toward antibiotics is high (antibiotic resistance is low) in the Arctic. Comparable results were obtained by Segawa et al. (2012) in which they reported the Antarctic region to have the least antibiotic resistance genes followed by the Arctic. In a similar study on bacteria isolated from the bottom sediments in the Wijdefjorden, Spitsbergen, Konieczna et al. (2011) observed that most of the bacterial isolates were sensitive to antibiotics such as cefoperazone, piperacillin, gentamycin, tobramycin, ciprofloxacin and ofloxacin. In the present study, it was observed that the most sensitive isolate (NA-9) and the most resistant isolate (MLB-31) both belonged to the genus Cryobacterium. This indicates that although the bacterial diversity in these microhabitats is limited, the strains differ widely in their antibiotic sensitivity characters. Segawa et al. (2012) and Sjolund et al. (2008) attribute the transfer of these antibiotic resistance characters in the Antarctic and the Arctic, to the airborne bacteria and migratory birds. Martinez (2012) on the other hand believes that the antibiotic resistance genes have been present for billions of years and have evolved over the years for detoxifying the original host from the antibiotic it produces. Whatever the case maybe, this adaptation strategy has helped the bacterial strains to overcome the antagonistic stresses and survive in all possible unique environments. Taxonomic analysis of the strains The phylogenetic tree constructed to determine the affiliation of the representative strains is shown in Fig. 3. The

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two Gram-negative groups (Pseudomonas and Polaromonas) belonging to the phylum Proteobacteria formed a distinct clade. One of the isolate, Flavobacterium, belonging to the phylum Bacteroidetes, was not considered during the phylogenetic tree construction because the sequence length was less than 1000 bp (Table 1). The 5 remaining Gram-positive groups were affiliated to phylum Actinobacteria. The isolates under this group showed close resemblance with the psychrophilic strains, reported from other parts of the world. An isolate of Cryobacterium from Crater lake, Antarctica and from Dongekemadi glacier, Qinghai-Tibetan Pleateau showed close affinity with Cryobacterium isolates. Leifsonia kafniensis, isolated from Kafni glacier, India and Leifsonia antarctica, isolated from Larsemann Hills, Antarctica showed close affinity with the Leifsonia isolate. The occurrence of related phylotypes in geographically diverse cold environments suggests that they possess comparable adaptation strategies by virtue of which they are able to tolerate and survive at low temperatures (Abyzov et al. 1998). These adaptation strategies include occurrence of pigments or polyunsaturated fatty acids or enzymes which are active at low temperature (Chintalapati et al. 2004; Shivaji et al. 2007). Distribution of the isolates in the three glaciers cryoconites The bacterial isolates from the cryoconites belonged to 8 genera namely, Pseudomonas, Polaromonas, Micrococcus, Subtercola, Agreia, Leifsonia, Cryobacterium, and Flavobacterium. Genera such as Pseudomonas, Flavobacterium, Cryobacterium are of common occurrence in cryoconite habitat and have been previously reported from the Antarctic cryoconites (Christner et al. 2003). Genera such as Pseudomonas and Flavobacterium have also been reported in the Alpine cryoconites (Lee et al. 2011). Other genera such as Polaromonas, Micrococcus, Subtercola and Agreia are cold-adapted taxa and have been reported earlier from Arctic habitats such as snow, permafrost and sea ice (Bakermans et al. 2003; Groudieva et al. 2004; Steven et al. 2007; Harding et al. 2011). Most of the species reported during this study (Cryobacterium psychrotolerans, C. psychrophilum, Leifsonia antarctica, Polaromonas naphthalenivorans, Pseudomonas ficuserectae, Micrococcus endophyticus and Subtercola frigoramans) have a GC content of [55 %. Only one species, Flavobacterium degerlachei, was reported with low GC content. Bacterial isolates with differential GC content was observed by Xiang et al. (2005) at Muztag Ata Glacier and by Zhang et al. (2008) in Puruogangri Ice Core. Xiang et al. (2005) attributed this discrepancy in GC content to the differences in bacterial deposition source and to nutrient availability


within the ice. The diversity indices indicate that when the three glaciers are compared VB has a greater diversity of cryoconite bacteria followed by ML and then AB. The bacteria at ML have higher evenness index as compared to the bacteria at the other two glacier sites. Carbon utilization ability Organic carbon compounds are particularly relevant for microbial metabolism in the cryoconite samples, as the range of carbon sources is limited to available organic and inorganic matter within the holes (Foreman et al. 2007). From the results of carbon utilization tests, it can be observed that several of the bacterial isolates from the cryoconite holes prefer monosaccharides, the simpler forms of carbohydrates, such as mannose, rhamnose and xylose, as carbon source. In similar studies on carbon utilization by bacteria in the surface sea waters of the Arctic Ocean, it was observed that the isolates utilized D-cellobiose and n-acetyl-D-glucosamine, which are common sources of carbon in the marine environment (Sala et al. 2010). Carbon utilization studies carried out with bacteria isolated from the marine beach on Southern Baltic coast, Poland also indicated a high percentage of mannose utilization (Mudryk et al. 2005). The isolates, however, were unable to utilize xylose. Studies in the Gdansk Deep region of Baltic sea also indicated that amongst the simple sugars tested mannose was best utilized followed by xylose (Donderski et al. 1998). Extracellular enzymatic activities Psychrophilic organisms due to their ability to produce cold active enzymes with potential applications in biotechnology, agriculture and medicine have attracted the attention of the scientific community (Feller and Gerday 2003). All the 21 representative strains tested showed activities of one or more enzymes (amylase, cellulase, lipase, urease, protease, or catalase) either at 4, 10 and (or) 20 °C (Supplementary Table 4). The ability of the bacterial strains, isolated from cryoconite holes, to produce different enzymes speaks about their ability to utilize the available organic matter, within the restricted environment, as source of carbon and nitrogen for their survival and growth. Previous study on substrate utilization by bacterial isolates from the surface waters of Arctic ocean revealed that carbohydrates and polyols play a major role in bacterial metabolism in the Arctic (Sala et al. 2008, 2010) and Antarctic environments (Sala et al. 2005). Cellulose and urea degradation by these isolates at low temperatures demonstrates their biotechnological potential. Prominent amongst the previous reports of extracellular enzyme activities by Arctic bacteria are Yu et al. (2009),

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Reddy et al. (2009) and Srinivas et al. (2009). While Srinivas et al. (2009) evaluated the amylase and lipase activities at different temperatures, Reddy et al. (2009) measured protease activity as well. Yu et al. (2009) extensively screened the bacterial isolates for production of protease, lipase, amylase, b-galactosidase and chitinase. This is the first report of screening cellulase, catalase and urease from cryoconite bacterial isolates. Fatty acid profiles Isolates of genus Cryobacterium and Micrococcus were subjected to fatty acid analysis. It was observed that in all the isolates the concentration of branched fatty acids was much higher than the saturated fatty acids. The most dominant fatty acids recorded in the isolates are anteiso-C15:0, anteiso-C17:0, iso-C15:0, iso-C16:0 and anteiso-C15:1 A. These results are in agreement with the earlier studies which indicated that unsaturated or branched fatty acids are common in coldadapted bacteria (Chintalapati et al. 2004; Reddy et al. 2009; Srinivas et al. 2009; Shivaji et al. 2013). In fact, the role of anteiso-C15:0 in the growth of Listeria monocytogenes at low temperature has been studied (Annous et al. 1997). They suggest that in order to grow at low temperatures bacterial cells undergo two modes of adaptation in their fatty acid composition; they shorten the length of fatty acid chain and alter the branching pattern from iso to anteiso. Fatty acids have a significant role to play in maintaining the membrane fluidity of the organisms and, therefore, are crucial for their continued survival at low temperatures (Nishida and Murata 1996). Cryoconite habitats often remain covered with ice during the long winter season. During the short summers when the overlying snow melts away, the cryoconites get exposed to the exterior atmosphere. To overcome the cold stress, the bacterial isolates growing in these cryoconite holes, may produce such branched forms of fatty acids.

Conclusions Cryoconite holes are important microcosms on the glacier where cold-adapted microbes flourish. The present study was focussed on characterizing the bacterial communities present in these cryoconite holes on the three Arctic glaciers through culture-based approach. Although the culture-based approach has its limitations of documenting incomplete diversity, it is an important technique for obtaining pure isolates for physiological and biochemical studies. To the best of our knowledge, this is the first report of culturable bacterial communities and their characterization from glacier cryoconites of High Arctic. The total and culturable bacterial counts indicate that the bacterial population in these holes is sizeable. 16S rRNA gene sequence analysis of the

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representative isolates identified them as belonging to 8 genera namely Pseudomonas, Polaromonas, Micrococcus, Subtercola, Agreia, Leifsonia, Cryobacterium, and Flavobacterium. These are some of the dominant genera known to occur in the colder habitats. The isolates showed varied response to antibiotics indicating that although the bacterial diversity in these microhabitats is limited, the strains differ widely in their antibiotic sensitivity characters. This adaptation strategy has helped the bacterial strains to overcome the antagonistic stresses and survive in all possible unique environments. Carbon utilization tests indicated that the bacterial isolates from cryoconite holes prefer simpler forms of carbohydrates, such as mannose, rhamnose and xylose, as carbon source. They are found to produce a variety of enzymes such as amylase, cellulase, lipase, urease, protease and catalase, at various incubation temperatures demonstrating their biotechnological potential. Fatty acid analysis indicates that the cryoconite bacterial isolates produce high amounts of branched fatty acids. The branched fatty acid and cold-adapted enzyme production are adaptation strategies to low temperature. The organisms have probably evolved these adaptation mechanisms for carrying out metabolic activities for growth, multiplication, survival and protection at low and even at sub-zero temperatures. These ecological adaptive strategies are of great interest and future studies need to be focussed on understanding their functioning at molecular level. Acknowledgments We would like to thank the Directors, BITS, NCAOR and ARI for facilities. PS acknowledges the DST, Government of India for the financial support (Project: SR/WOS-A/LS-68/ 2009). SMS is thankful to Ms Simintani Naik, Mr Masaharu Tsuji and Mr Jakub Zˇa`rsky´ for technical help.

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Draft genome of Cryobacterium sp. MLB-32, an obligate psychrophile from glacier cryoconite holes of high Arctic.

Rhodotorula svalbardensis sp. nov., a novel yeast species isolated from cryoconite holes of Ny-Ålesund, Arctic.

Potential sources of bacteria colonizing the cryoconite of an Alpine glacier.

A new approach for discovering cold-active enzymes in a cell mixture of pure-cultured bacteria.

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.

Polychlorinated Biphenyl (PCB)-Degrading Potential of Microbes Present in a Cryoconite of Jamtalferner Glacier.

Can the Bacterial Community of a High Arctic Glacier Surface Escape Viral Control?

Bacterial diversity of Drass, cold desert in Western Himalaya, and its comparison with Antarctic and Arctic.

Diversity of immune strategies explained by adaptation to pathogen statistics.

Diversity and role of plasmids in adaptation of bacteria inhabiting the Lubin copper mine in Poland, an environment rich in heavy metals.

Effect of cold adaptation on activities of relevant enzymes and antioxidant system in rats.

Cold adaptation of microorganisms.

Cold destabilisation of enzymes.

VEGF189 expression is highly related to adaptation of the plateau pika (Ochotona curzoniae) inhabiting high altitudes.

An active atmospheric methane sink in high Arctic mineral cryosols.

Culturable diversity and functional annotation of psychrotrophic bacteria from cold desert of Leh Ladakh (India).

Soil fungal community development in a high Arctic glacier foreland follows a directional replacement model, with a mid-successional diversity maximum.

Rich and cold: diversity, distribution and drivers of fungal communities in patterned-ground ecosystems of the North American Arctic.

Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden, Svalbard (High Arctic).

Considering the Specific Impact of Harsh Conditions and Oil Weathering on Diversity, Adaptation, and Activity of Hydrocarbon-Degrading Bacteria in Strategies of Bioremediation of Harsh Oily-Polluted Soils.

Mechanisms of adaptation to cold.

Spring thaw ionic pulses boost nutrient availability and microbial growth in entombed Antarctic Dry Valley cryoconite holes.

Adaptational properties and applications of cold-active lipases from psychrophilic bacteria.

High-throughput sequencing-based analysis of endogenetic fungal communities inhabiting the Chinese Cordyceps reveals unexpectedly high fungal diversity.