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Antarctic ice core samples: culturable bacterial diversity
Research in Microbiology 164 (2013) 70e82 www.elsevier.com/locate/resmic
Antarctic ice core samples: culturable bacterial diversity Sisinthy Shivaji a,*, Zareena Begum a, Singireesu Soma Shiva Nageswara Rao a, Puram V. Vishnu Vardhan Reddy a, Poorna Manasa a, Buddi Sailaja a, Mambatta S. Prathiba a, Meloth Thamban b, Kottekkatu P. Krishnan b, Shiv M. Singh b, Tanuku N.R. Srinivas a a
b
Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India National Center for Antarctic and Ocean Research, Headland Sada, Vasco da Gama, Goa, India Received 24 October 2011; accepted 4 September 2012 Available online 4 October 2012
Abstract Culturable bacterial abundance at 11 different depths of a 50.26 m ice core from the Tallaksenvarden Nunatak, Antarctica, varied from 0.02 to 5.8 103 CFU ml1 of the melt water. A total of 138 bacterial strains were recovered from the 11 different depths of the ice core. Based on 16S rRNA gene sequence analyses, the 138 isolates could be categorized into 25 phylotypes belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. All isolates had 16S rRNA sequences similar to previously determined sequences (97.2e100%). No correlation was observed in the distribution of the isolates at the various depths either at the phylum, genus or species level. The 25 phylotypes varied in growth temperature range, tolerance to NaCl, growth pH range and ability to produce eight different extracellular enzymes at either 4 or 18 C. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a significant proportion of the total fatty acid composition. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: 16S rRNA gene; Actinobacteria; Bacteroidetes; Firmicutes; Proteobacteria; Phenotypic characteristics; Extracellular enzymatic activities; Fatty acid profiles
1. Introduction Ice cores offer a unique opportunity to correlate microbial diversity with age and prevailing chemical conditions in ice cores (Castello and Rogers, 2005). Studies on ice cores from the Malan glacier, the Muztag Ata glacier and the Puruogangri glacier (Zhang et al., 2003; Xiang et al., 2005) established that bacterial abundance positively correlated with dust within the ice core (Zhang et al., 2006a). Furthermore, the phylogenetic diversity of bacteria in the ice cores revealed a high abundance
* Corresponding author. Tel.: þ91 40 27192504; fax: þ91 40 27160311. E-mail addresses: [email protected] (S. Shivaji), [email protected] (Z. Begum), [email protected] (S.S. Shiva Nageswara Rao), [email protected] (P.V. Vishnu Vardhan Reddy), [email protected] (P. Manasa), [email protected] (B. Sailaja), [email protected] (M.S. Prathiba), [email protected] (M. Thamban), [email protected] (K.P. Krishnan), [email protected] (S.M. Singh), [email protected] (T.N.R. Srinivas).
of members of the phyla Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes in Tibetan ice cores, the Puruogangri Ice core,the Muztag Ata glacier ice core and the Deep Greenland glacier ice core (Miteva et al., 2004; Xiang et al., 2005; Zhang et al., 2002, 2003, 2008). In some ice cores, as in the Greenland glacier, in addition to aerobic bacteria, anaerobic bacteria related to Thermus, Bacteroides, Eubacterium and Clostridium groups were also found (Miteva et al., 2004; Moore et al., 1998). Studies on species diversity, genetics and physiological differences in ice core isolates is crucial, since bacteria with nearly or fully matching 16S rRNA gene sequences exhibit distinct physiological capabilities (Moore et al., 1998). Despite the above studies, very few studies have been carried out on ice cores from Antarctica (Abyzov et al., 2001; Christner et al., 2001, 2002; Foreman et al., 2011; Karl et al., 1999). This is all the more important, since psychrophilic bacteria at the species level need to be documented and studied to a greater extent so as to be able to exploit their
0923-2508/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.resmic.2012.09.001
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
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potential for biotechnological application (Feller and Gerday, 2003). Some psychrophilic bacteria are able to produce omega-3 polyunsaturated fatty acids which are of dietary significance (Nichols et al., 1995). Psychrophilic bacteria are also potential sources of novel pigments (they can be used as food additives) and cold-active enzymes for industrial application (Feller and Gerday, 2003). The goal of the present study was to establish bacterial abundance and viable bacterial diversity in an ice core sample collected from the continental ice sheet at central Dronning Maud Land, Antarctica. In addition, these bacteria were also used for bioprospecting for fatty acids and cold-active enzymes.
Research, Goa, India, in a cold room facility (20 C). Prior to chemical and microbiological studies, the surface of the ice core was scraped with a sterile microtome blade and then a slice of ice from a particular depth was cut and separated using a sterile band-saw machine (Table 1). Only the inner unexposed core of the sample was used for microbiological analysis. Utmost care was taken to minimize contamination during sectioning and subsequent analysis. The core samples were melted in a class 100 clean room before analysis. The age of the ice core was estimated according to the method employed earlier (Thamban et al., 2006).
2. Materials and methods
The melt water from the ice core sample was filtered through a 0.2 mm filter. Subsequently, the filter was placed on Antarctic bacterial medium (ABM) plates [peptone (0.5%, w/v), yeast extract (0.2%, w/v) and agar (2%, w/v)] and incubated at 10 C for 15 days. Different morphotypes that appeared were purified and maintained on ABM plates.
2.1. Sampling site and sampling method An ice core (IND-22/B4) of 50.26 m was recovered from the Tallaksenvarden nunatak located in central Dronning Maud Land (70 51.30 S and 11 32.20 E, elevation 680 m), Antarctica (Fig. 1), during the 22nd Indian Scientific Expedition to Antarctica. The annual mean temperature and snow accumulation reported at this site were w20 C and w170 kg m2 a1 respectively. Ice core drilling was carried out during the austral summer (FebruaryeMarch) of 2003 using an electromechanical drilling system (diameter w10 cm, type D-2; GeoTecs Ltd., Japan) and the retrieved ice core samples were labeled, packed in polythene containers and shipped to India in a deep-freezer at 20 C. Subsequently, the samples were archived in polypropylene insulated containers at the National Center for Antarctic and Ocean
2.2. Isolation and culturing of bacteria
2.3. Characterization of bacterial strains Colony characteristics were observed with the help of a magnifying lens. Growth at different temperatures, pH and salt concentrations were carried out on ABM plates. Extracellular enzymatic activities of amylase, lipase and proteases were checked by streaking the culture on ABM plates supplemented with 0.2% soluble starch or 1% Tween-60 along with 0.01% CaCl2 or 0.3% casein, respectively, incubated at 4 C and 18 C for 5e10 days. Gelatinase activity was checked by inoculating the culture in ABM broth supplemented with 10% gelatin and incubating the plates at 4 C and 18 C for 5e10 days. DNase, esculinase and urease activities were checked by streaking the culture on DNase test agar (M1041, HIMEDIA, Hyderabad, India), esculin agar (M1386, HIMEDIA, Hyderabad, India) or urea agar base (M112, HIMEDIA, Hyderabad, India) and by subsequently incubating the plates at 4 C and 18 C for 5e10 days. Catalase activity was tested by adding hydrogen peroxide to the culture taken on a slide. Fatty acid methyl esters were prepared and analyzed as previously described (Shivaji et al., 2007) using cultures grown at 18 C. 2.4. Tolerance to freezing and freeze-thaw
Fig. 1. Ice core sample collection point (IND-22/B4) in the Dronning Maud Land, Antarctica.
Tolerance of the bacteria to freezing and freeze-thaw was done by previously described methods (Sleight et al., 2006; Walker et al., 2006). Six cultures were selected based on their growth temperature range [AIC 11.11 (4e18 C), AIC 5.21 and AIC 5.24 (4e30 C) and AIC 3.7, AIC 5.1 and AIC 11.14 (4e37 C)] and grown in ABM broth for 5 days till they reached stationary phase. Then, 1 ml of the culture was taken and centrifuged at 8000 rpm at room temperature for 10 min. The cell pellet was suspended in distilled water mixed properly and the tube was placed in a 20 C freezer. Simultaneously, 18 tubes were prepared and 15 of them were frozen at
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Table 1 Bacterial abundance and physical characteristics at the 11 different depths (AIC-1 to AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica. CFU (103)/ml
Number of morpho-types
Strain numbers
4.8 0.3 4.0 0.9
10 13
17.72
3.2 1.0
10
1859
19.06
3.1 0.4
12
20.35e20.45
1814
19.22
3.3 0.4
10
6.4
25.11e25.21
1772
17.73
4.5 1.0
11
AIC7
6.5
30.30e30.4
1723
15.47
5.8 0.6
25
8
AIC8
6.4
34.88e34.98
1680
17.90
4.8 0.7
12
9
AIC9
6.4
40.08e40.18
1631
17.76
3.8 1.0
16
10
AIC10
6.4
44.88e44.98
1586
18.11
1.7 0.3
10
11
AIC11
6.4
50.16e50.26
1535
19.31
0.02 0.001
9
AIC1e1 to 1e6; 1e8; 1e25; 1e26; 1e29 AIC2e1; 2e5; 2e7 to 2e11; 2e13 to 2e15; 2e22; 2e27; 2e28 AIC3e7 to 3e10; 3e12B; 3e16; 3e19 to 3e22 AIC4e1; 4e2; 4e5 to 4e8; 4e10 to 4e14; 4e18 AIC5e1; 5e7 to 5e10; 5e18; 5e21; 5e24; 5e25; 5e28 AIC6e1; 6e4; 6e5; 6e11A; 6e11B; 6e14; 6e24; 6e26; 6e27; 6e29; 6e30 AIC7e2; 7e5 to 7e8; 7e10 to 7e18; 7e20; 7e21; 7e23 to 7e31 AIC8e2 to 8e6; 8e9; 8e14; 8e26 to 8e28; 8e31; 8e32 AIC9e1 to 9e6; 9e8; 9e10; 9e12 to 9e17; 9e20; 9e26 AIC10e9; 10e10; 10e13 to 10e16; 10e21; 10e25; 10e30; 10e31 AIC11e10; 11e11; 11e14; 11e19; 11e20; 11e23; 11e24; 11e28; 11e30
Sl.no.
Sample ID
pH
1 2
AIC1 AIC2
7 6.8
3
AIC3
4
Depth (m)
Age (AD)
Ice core surface temperature ( C)
0.25e0.35 5.06e5.16
1993 1940
18.85 21.57
6.7
10.36e10.46
1892
AIC4
6.5
15.26e15.36
5
AIC5
6.4
6
AIC6
7
20 C. Subsequently, at each time point (5, 9, 12, 14 and 15 days of freezing), 3 tubes were removed from the freezer, thawed for 1.5 h at 22 C and then 100 ml of the thawed culture was serially diluted in 900 ml of 0.9% saline. The diluted culture (100 ml of the suspension) was plated on TSA plates and incubated at 18 C for 3 days. Colony counts were recorded in triplicate, the average calculated and used for determining the percentage of cell survival, considering the cell count on day 0 as 100%. The remaining 3 tubes that were not frozen served as the zero time point control and were plated as above on day 0. Tolerance of the six cultures to freeze-thaw cycles was also tested. In these experiments, one ml of the culture in triplicate was prepared as above and frozen at 20 C. At the respective cycle number (0, 3, 7, 9, 11, 13 and 20), the cultures in triplicate were thawed for 1 h and then 100 ml of the thawed culture was serially diluted, plated and processed for viable counts as above. Each freeze-thaw cycle consisted of 1 h freezing at 20 C and thawing for 1.5 h at 22 C.
phylogenetic trees were constructed using the maximum likelihood (ML) method (Anil Kumar et al., 2010).
2.5. PCR amplification of 16S rRNA gene sequencing and phylogenetic analysis
3.2. Bacterial count
The 16S rRNA gene was amplified using pA (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and pH* (50 -AAG GAG GTG ATC CAG CCG CA-30 ) as previously described (Shivaji et al., 2006) and the resulting amplicons were sequenced using different internal primers (Lane, 1991). The 16S rRNA gene sequences were then subjected to BLAST sequence similarity search (Altschul et al., 1990) to identify the nearest taxa. Subsequently, the sequences were aligned with the sequences from the nearest taxa using the CLUSTAL_X program and
2.6. Nucleotide sequence accession numbers All 16S rRNA gene sequences of the strains were deposited in GenBank with accession numbers JF970575 to JF970601. 3. Results and discussion 3.1. Characteristics of ice core samples collected from Dronning Maud Land, Antarctica The pH in the ice core sample was neutral at the surface, but at all other depths up to 50 m, the pH was slightly acidic. Further, the estimated deposition date of the ice core ranged from 1993 AD for the surface 0.25 m layer to 1535 AD at a depth of 50.26 m. The ice core surface temperature varied at different depths of the ice core (Table 1).
The culturable bacterial count (colony-forming units, CFUs) in the Antarctic ice core varied from 0.02 to 5.8 103 CFU ml1 of the ice core melt water (Table 1). The minimum number was recorded at a depth of 50.16 m (Table 1). This is similar to that observed for Puruogangri ice core melt water, where CFU fluctuated from 0 to 760 CFU ml1 (Zhang et al., 2008). Earlier studies also indicated that the estimates of microbes in glacial ice differed widely with the geographic location, and the number varied from less than one viable cell ml1 in polar ice (Abyzov et al., 1982) to
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6 107 cells ml1 in a Greenland ice core (Sheridan et al., 2003). Further, the CFU ml1 of the ice core melt water did not reveal any appreciable tendency to change with increasing depth, thus implying that the microorganisms were episodically deposited in the glacier (Zhang et al., 2008). 3.3. Viable bacteria A total of 138 bacterial strains were recovered from the 11 different depths of the ice core (Tables 1 and 2). These 138 strains, based on similarity criteria of 97% at the 16S rRNA gene sequence level, could be categorized into 25 phylotypes (Table 2). Each group represented a different taxon (Table 2),
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and since 97% sequence similarity was used as the cut-off, the groups varied with the nearest phylogenetic neighbor at the species level. Based on Gram staining, 11 groups were Gram-negative and 16 were Gram-positive. Out of the 25 phylotypes, 11 were common to two or more depths of the ice core and the most common phylotype was Pseudomonas azotoformans, which was shared by 7 out of the 11 depths of the ice core (Supplementary Table 4). 3.4. Characteristics of the isolated strains A representative from each of the 25 phylotypes was chosen to study some of their phenotypic (NaCl tolerance,
Table 2 Identification of the 138 bacterial strains isolated from the 11 different depths (AIC-1 to AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica based on BLAST analysis of the 16S rRNAa gene sequences. Sl.No.
Strain number
Nearest phylogenetic neighbor and 16S rRNA gene sequence similarity (%)
AIC5e18 AIC5e28 AIC5e24, 5e25 AIC1e8, 5e9, 5e10, 9e1 to 6, 9e8, 11e23, 11e28, 11e30 AIC8e9, 1e26, 1e29, 2e11, 5e21, 6e26
Arthrobacter rhombi F.98.3HR.69T Y15885, (99.2) Brevibacterium antiquum VKM Ac-2118T AY243344, (98.5) Rhodococcus cercidiphylli YIM 65003T EU325542, (99.1e99.4) Rhodococcus fascians DSM 20669T X79186, (99.4e99.9)
Bacillus psychrodurans DSM 11713T AJ277984, (99.4) Bacillus stratosphericus 41KF2aT AJ831841, (99.9) Brevibacillus centrosporus DSM 8445T AB112719, (99.8) Brevibacillus panacihumi DCY35T EU383033, (97.9) Paenibacillus humicus PC-147T AM411528, (98.7e99.0) Paenibacillus peoriae DSM 8320T AJ320494, (99.1e99.7)
12 13 14
AIC11e10 AIC10e21 AIC3e19 AIC3e7 AIC2e1, 2e5, 2e7, 2e22, 2e27, 2e28 AIC4e2, 4e5, 4e6, 4e7, 5e1, 5e7, 5e8, 11e19, 11e20, 11e24 AIC3e20, 3e21, 3e22 AIC11e11 AIC3e16
Paenibacillus xylanexedens B22aT EU558281, (99.0e99.4) Sporosarcina globispora DSM 4T X68415, (99.6) Staphylococcus sciuri subsp. sciuri DSM 20345T AJ421446, (99.6)
Bacteroidetes 15 16
AIC6e11A AIC6e5
Flavobacterium pectinovorum DSM 6368T AM230490, (98.2) Sphingobacterium anhuiense CW186T EU364817, (99.9)
AIC6e11B, 2e14, 2e15, 6e1 AIC10e25, 10e30, 10e31, 10e9, 9e26, 9e14, 9e10 AIC1e1, 1e2, 1e4 to 1e6, 1e25, 2e8 to 2e10, 2e13, 4e10, 6e24, 6e27, 6e29, 7e2, 7e5 to 7e8, 7e10, 7e11, 7e13 to 7e18, 7e20, 7e21, 7e23 to 7e31, 8e2 to 8e6, 8e14, 8e26 to 8e28, 8e31, 8e32, 9e12, 9e15 to 9e17, 9e20 AIC6e14 AIC7e12, 1e3 AIC11e14, 9e13 AIC10e10, 10e14, 10e16 AIC3e8, 3e9, 3e10, 3e12B, 10e13, 10e15 AIC4e1, 4e8, 4e11, 4e12, 4e13, 4e14, 4e18, 6e4, 6e30
Janthinobacterium lividum DSM 1522T Y08846, (99.6e99.7) Naxibacter haematophilus CCUG 38318T AM774589, (98.5e98.9)
Actinobacteria 1 2 3 4 5 Firmicutes 6 7 8 9 10 11
Proteobacteria 17 18 19
20 21 22 23 24 25
Rhodococcus qingshengii djl-6T DQ090961, (99.9e100.0)
Pseudomonas azotoformans IAM1603T D84009, (97.5e99.9)
Pseudomonas brenneri CFML 97-391T AF268968, (99.5) Pseudomonas poae DSM 14936T AJ492829, (99.3e99.9) Pseudomonas stutzeri CCUG 11256T U26262, (100.0) Sphingomonas aerolata NW12T AJ429240, (99.2) Sphingomonas faeni MA-olkiT AJ429239, (97.2e99.4) Stenotrophomonas rhizophila e-p10T AJ293463, (97.5e100.0)
a The 16S rRNA gene sequences of all 138 strains were obtained and subjected to BLAST and phylogenetic analyses. Based on phylogenetic analyses, the 138 strains were categorized into 25 groups and representatives of each group (indicated in bold) were submitted to EMBL (JF970575 to JF970601).
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temperature and pH range), biochemical (amylase, caseinage, catalase, DNase, esculinase, gelatinase, lipase and urease activities) and chemical (fatty acid composition) characteristics (Supplementary Tables 1e3). The 25 representative strains were either psychrophilic or psychrotolerant. The psychrophilic strains were able to grow between 4 and 18 C (5 strains), whereas the psychrotolerant strains were capable of growth at between 4 and 30 C (10 strains) or 4 and 37 C (10 strains) (Supplementary Table 1). Psychrotolerant bacteria capable of growing between 4 and 37 C were reported earlier (Srinivas et al., 2009; Reddy et al., 2009). The present observation that psychrotolerant bacteria (80.0%) are predominant, followed by the psychrophiles (20.0%), is in accordance with the earlier study of Xiang et al. (2005), who observed that bacteria isolated from the Muztag Ata glacier at increasing depths were predominantly psychrotolerant (82%), followed by psychrophiles (11%) and then mesophiles (7%). The Muztag Ata glacier ice isolates and the current isolates had temperature growth profiles consistent with adaptation to growth under cold growth conditions, and most likely were transferred by wind or snow from local ecosystems onto the glacier’s surface (Xiang et al., 2005). All strains were able to grow without NaCl in the medium, and tolerance to NaCl varied from 0.5 to 2 M (Supplementary Table 1). One of the most interesting results is that a high percentage of the isolates could grow in a very narrow pH range, with strains either growing only between pH 6e8, 6e9 and 7e9 (Supplementary Table 1). Strains having a narrow pH range for growth had been reported earlier (Srinivas et al., 2009; Reddy et al., 2009). 3.5. Tolerance of bacterial strains to freezing and freeze-thaw cycles Six bacterial strains were selected for the experiment based on their growth temperature range [AIC-11.11 (4e18 C), AIC-5.21 and AIC-5.24 (4e30 C) and AIC-3.7, AIC-5.1 and AIC-11.14 (4e37 C)] which varied from 4 to 18 to 4e30 and 4e37 C. For up to 5 days at 20 C, none of the cultures showed any appreciable decrease in survival, but by day 9 survival decreased and it was most pronounced in AIC-5.21, AIC-3.7, AIC-5.1 and AIC-11.14 (Fig. 3a). Subsequently, by day 15, survival of all cultures decreased to 10%. Culture AIC-11.11 Sporosarcina globispora, with a growth temperature range of 4e18 C, appeared to be the most tolerant to freezing (Fig. 3a) compared to the other cultures. Compared to continuous freezing, the above six cultures showed more resistance to freeze thaw cycles. Strains AIC3e7, AIC5e21, AIC11e14 and AIC11e11 showed >10% survival even after 20 freeze-thaw cycles. But AIC5e1 and AIC5e24 showed drastic decreases in growth after cycle 3 (Fig. 3b). This ability to resist freeze-thaw cycles is an adaptive feature required for survival in ice cores. However, the present study did not find any correlation between growth temperature range and percent survival after freezing or freeze thaw conditions.
The fatty acid profile data also support the hypothesis that these isolates adapt to a cold environment by possessing more branched and unsaturated fatty acids (analyzed by growing the cultures at 18 C) (Supplementary Tables 2 and 3). 3.6. Taxonomic analysis of the 25 representative strains The phylogenetic trees constructed to determine the affiliation of the representative strains of the 25 groups are shown in Fig. 2a and b. Among the 11 Gram-negative groups, 9 belonged to the phylum Proteobacteria and two to the phylum Bacteroidetes (Table 2 and Supplementary Table 4). The nearest phylogenetic neighbors of 9 of the 11 Gram-negative representative strains had been earlier isolated from cold habitats and were reported to be either psychrophilic or psychrotolerant, and include Janthinobacterium lividum (Reddy et al., 2009), P. azotoformans (Srinivas et al., 2009), Pseudomonas brenneri and Pseudomonas stutzeri (Srinivas et al., 2009), Pseudomonas poae, Sphingobacterium anhuiense, Sphingomonas aerolata, Sphingomonas faeni and Stenotrophomonas rhizophila. Only two strains, AIC6e11 and AIC9e14, related to Flavobacterium pectinovorum and Naxibacter haematophilus, respectively, had not been reported earlier from any cold habitat. Among the 16 Gram-positive groups, 5 were affiliated with the phylum Actinobacteria and the nearest phylogenetic neighbors were Arthrobacter rhombi (Osorio et al., 1999), Brevibacterium antiquum, Rhodococcus cercidiphylli, Rhodococcus fascians and Rhodococcus qingshengii (Srinivas et al., 2009) (Table 2 and Supplementary Table 4). Psychrophilic and psychrotolerant strains of the above nearest phylogenetic neighbors were earlier isolated from cold habitats like Greenland, permafrost samples and the Arctic. The remaining 11 Gram-positive groups were affiliated with the phylum Firmicutes and represented by genera/species of Bacillus, Brevibacillus, Lysinibacillus fusiformis, Paenibacillus, S. globispora and Staphylococcus sciuri (Table 2 and Supplementary Table 4). Only a few of the phylogenetic neighbors of the 11 groups affiliated with Firmicutes were isolated from cold habitats like the Alaskan tundra and the stratosphere, and they include Bacillus stratosphericus (Shivaji et al., 2006) and Paenibacillus xylanexedens. Four other psychrotolerant strains, namely Bacillus psychrodurans, Brevibacillus centrosporus, Paenibacillus peoriae and S. globispora, have not been reported from any cold habitat. Thus, the results indicate that most of the 25 representative strains are affiliated with species/genera from cold habitats except for those representative strains affiliated with the genera Lysinibacillus, Staphylococcus and Naxibacter. The occurrence of related phylotypes in geographically diverse cold environments suggests that they are tolerant to a cold environment and possess similar strategies to survive freezing and remain active at low temperatures (Abyzov et al., 1998). The ability of psychrophilic and psychrotolerant bacteria to adapt to cold conditions may be ascribed to the occurrence of pigments or polyunsaturated fatty acids, and with enzymes that are active
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82 Fig. 2. Phylogenetic trees based on 16S rRNA gene sequences showing the relationship of Gram-positive (a) and Gram-negative (b) strains obtained from the Antarctic ice core sample collected from the continental ice sheet at central Dronning Maud Land, Antarctica, with their nearest phylogenetic relatives. Phylogenetic trees were constructed by the by maximum likelihood method. Numbers at nodes are bootstrap values (50). Aquifex pyrophilus Kol5aT (M83548) was used as an outgroup. The bar represents 0.02 substitutions per alignment position in (a) and (b).
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Fig. 3. Tolerance of bacteria to freezing (a) and freeze-thaw cycles (b). (a) Survival of 6 bacterial taxa up to a period of 15 days at 20 C was monitored. (b) Survival of the same 6 bacterial taxa to 20 cycles of freeze-thaw was monitored. Each freeze-thaw cycle consisted of 1 h freezing at 20 C and thawing at 22 C for 1.5 h. The bacterial taxa studied were (O) ¼ AIC 5:1; (,) ¼ AIC 3:7; (D) ¼ AIC 11:11; (C) ¼ AIC 5:24; (-) ¼ AIC 5:21; (:) ¼ AIC 11:14.
at low temperature (Chintalapati et al., 2004; Shivaji et al., 2007). Most of the isolates (79%) were found to be non-sporeformers and only 29 out of 138 isolates produced spores and belonged to the genera Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus and Sporosarcina. This is in accordance with the earlier observations that non-spore-forming bacteria are more abundant in glacial ice (Shafaat and Ponce, 2006). The abundance of non-spore formers in ice has been attributed to their ability to survive for extended periods of time in cold conditions because their DNA degrades far more slowly, and DNA repair is probably more efficient than that in spore formers (Johnson et al., 2007). 3.7. Vertical distribution of the isolates in the ice core The 138 isolates from the 11 depths of the ice core belonged to four different phyla: Actinobacteria (high G þ C), Firmicutes (low G þ C), Bacteroidetes and Proteobacteria (Table 2). Thus, current data support earlier data showing that members of the phyla Proteobacteria, Bacteroidetes and Actinobacteria are common in most cold habitats (Cheng and Foght, 2007; Neufeld et al., 2004; Shivaji et al., 2011; Steven et al., 2007; Srinivas et al., 2009; Reddy et al., 2009). In the
present study, the most frequently isolated bacteria belonged to the phylum Proteobacteria (61.5%) and were detected at all depths except in AIC-5 (20.35 m). The most predominant taxon was P. azotoformans, which was isolated from 7 depths of the ice core (Table 2 and Supplementary Table 4). Isolates affiliated with the phylum Firmicutes were the next most abundant (21%); they exhibited discontinuous distribution along the depth of the ice core and were not obtained on the surface or at depths corresponding to AIC-6 to AIC-9 (25.11e40.08 m respectively) (Table 2 and Supplementary Table 4). The dominant taxon among Firmicutes was Paenibacillus peoriae. Isolates affiliated with the phylum Actinobacteria constituted 16.1% of the total isolates and were represented by five taxon, all present in AIC-5 but absent in AIC-3, AIC-4, AIC-7 and AIC-10 (Table 2 and Supplementary Table 4). The phylum Bacteroidetes was the least represented (1.4%) and was present only in AIC-6 (25.11 m) (Table 2 and Supplementary Table 4). AIC-6 showed maximum diversity represented by 7 different species and AIC-7 and AIC-8 showed minimal diversity represented by only two species. Only P. azotoformans and Rhodococcus quingshengii were common to 5 depths of the ice core. The results indicate that distribution of taxa did not exhibit a specific pattern of distribution along the length of the ice core. For instance, Actinobacteria and Proteobacteria were present in AIC-1, AIC-8 and AIC-9, Actinobacteria, Firmicutes and Proteobacteria in AIC-2 and AIC-11, Firmicutes and Proteobacteria in AIC-3, AIC-4 and AIC-10, Actinobacteria and Firmicutes in AIC-5, Actinobacteria, Bacteroidetes and Proteobacteria in AIC-6 and only Proteobacteria in AIC-7 (Table 2 and Supplementary Table 4). Similar differential distribution of high G þ C, low G þ C, Bacteroidetes and Proteobacteria have been observed in ice cores from the Muztag Ata glacier (Xiang et al., 2005) and in the Puruogangri ice core (Zhang et al., 2008). Xiang et al. (2005) attributed the differential distribution to differences in bacteria deposited serendipitously on the glacier’s surface by wind and snowfall, and nutrient availability within the ice (Xiang et al., 2005). Proteobacteria, high G þ C, low G þ C and Bacteroidetes group bacteria were earlier also isolated from a Vostok (Antarctica) ice core (Christner et al., 2001) and the Siple glacier, Antarctica (Christner et al., 2001). 3.8. Extracelluar 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 25 representative strains showed activities of one or more enzymes (amylase, caseinase, catalase, DNase, esculinase, gelatinase, lipase or urease) either at 4 C and (or) 18 C (Supplementary Table 1). Only 3 strains (AIC2e15, AIC3e20 and AIC4e13) showed 5 out of 8 different enzymatic activities (Supplementary Table 1). Furthermore, 19 of the 25 representative strains produced extracellular enzymes at 4 C (Supplementary Table 1).
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
3.9. Fatty acid profiles In psychrophilic and psychrotolerant bacteria (all phylotypes), branched fatty acids varied in composition from a minimum of 0.3%, as in AIC2e15, to a maximum of 92.5% as in AIC1e3 (Supplementary Tables 2 and 3). In AIC2e15, the low % of branched fatty acids was compensated for by a high proportion of unsaturated fatty acids (74.1%) (Supplementary Table 2). The saturated fatty acid composition in all isolates varied in composition from a minimum of 2.0%, as in AIC5e24, to a maximum of 59.3, as in AIC5e1 (Supplementary Tables 2 and 3). These results are in accordance with earlier studies which indicated that unsaturated and branched (including the iso- and anteiso-) fatty acids are common in psychrophilic and psychrotolerant bacteria (Chintalapati et al., 2004) and play an important role in maintaining functional membrane fluidity crucial for continued survival at low temperatures (Nishida and Murata, 1996). Thus, it appears that psychrophilic and psychrotolerant bacteria from ice cores adapt to the cold environment by preferentially synthesizing unsaturated and branched fatty acids. 3.10. Conclusions Bacterial abundance in an ice core sample collected from the continental ice sheet at central Dronning Maud Land,
77
Antarctica, was observed to be similar to that estimated in the ice core samples from other such habitats (Abyzov et al., 1982; Sheridan et al., 2003). Present data represent only viable bacteria from the ice core and thus reveal only a part of the microbiome. Thus, there is a need to carry out more detailed studies to address the total biodiversity of the ice core using the metagenome approach. The ability of the microorganisms to survive prolonged periods of freezing and multiple cycles of freeze-thaw reflect the ability of psychrophiles and psychrotolerant bacteria to adapt to low temperature conditions. Bioprospecting studies indicated that these isolates could be used as a bioresource for the generation of psychrophilic enzymes. Earlier studies on culturable bacteria suggested that distribution of isolates at different depths in ice cores may correlate with specific past climatic as well as environmental conditions, such as the monsoon (Petit et al., 1999; Thompson et al., 2000; Zhang et al., 2006b), temperature, nutrient availability (Petit et al., 1999; Thompson et al., 2000; Xiang et al., 2005) and dust content (Zhang et al., 2002). Thus, to gain better insight into bacterial diversity in ice cores with respect to temporal and spatial variations, more comprehensive studies would need to be carried out using multiple ice cores, employing both culture- and molecularbased approaches (Zhang et al., 2008) and undertaking seasonal studies so as to understand the dynamics of deposition of organisms in ice.
Supplementary Table 1 Physiological characteristics of bacterial strains representative of the 25 bacterial groups isolated from the Antarctic ice core. Serial Strain ID number
Growth characteristics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
4e30 4e18 4e37 4e37 4e37 4e37 4e37 4e37 4e37 4e37 4e30 4e30 4e30 4e18 4e30 4e30 4e30 4e30 4e18 4e30 4e37 4e18 4e18 4e37 4e30
AIC1e3 AIC2e15 AIC2e22 AIC3e7 AIC3e9 AIC3e16 AIC3e19 AIC3e20 AIC4e13 AIC5e1 AIC5e18 AIC5e21 AIC5e24 AIC5e28 AIC6e5 AIC6e11 AIC6e14 AIC8e6 AIC9e14 AIC10e14 AIC10e21 AIC11e10 AIC11e11 AIC11e14 AIC11e28
Enzyme activities
pH range Amylase Esculinase Gelatinase Urease Casei-nase Cata-lase DNase Lipase Temperature NaCl tolerance range ( C) 4 C 18 C 4 C 18 C 4 C 18 C 4 C 18 C 18 C 18 C 18 C 18 C (M) 1 0.5 0 2 0 2 0 0.5 0.5 0.5 1 1 1 2 0.5 0.5 2 1 1 1.5 0.5 0.5 0.5 0.5 1
6e8 6e8 6e8 6e9 6e8 6e9 6e8 6e8 6e9 6e9 6e9 6e9 6e9 6e9 6e8 6e9 6e9 6e8 6e11 6e10 6e9 8e9 7e9 6e10 6e9
e e e e e e e þ e e e e e e e e e e e e e e e e e
e e þ e e e e e e þ e e þ e e e e e e e e e e e e
e þþþ þ e e þþþ e þþþ þþþ e þ e e e þþþ þþþ þþ e e e þþþ e e þþþ e
e þþ þþ þ e þþþ e þþþ þþþ þþþ þ þ e e þ þþ þ e þ e þþ e e þþ e
e e e e e e e e e e e e e e e e e e e e þ e e þ e
þ, Weakly positive; þþ, moderately positive; þþþ, highly positive; , negative; x, no growth.
þ þ e e þ e e e þ e e e e e e e e þ e e þ e e e e
e þ e þþþ e e þ e e e þþþ þþþ þþþ þþþ þþþ e e e þþþ e e þþþ þþþ þþþ e
þ e e þþþ e e e e þ e þþþ þþþ þþ þþþ þþþ þ e þ þþþ þþ e þþþ þþþ þþþ e
e e e e e e e þ e e e e e e e e e e e e e e e e e
þ þ w þ þ þ þ þ þ e þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ e e e e e e e e e e e e e e e e þ e e e e e e e
þþ þþ þþ e e e e þ þþ e þ e e e þþþ þþ e þ e þþþ þþ þ e e þþþ
78
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
Supplementary Table 2 Fatty acid profiles of the 11 representative Gram-negative bacterial strains among the 25 bacterial groups isolated from the 8 different depths (AIC-1 to 4; AIC-6; AIC-9e11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica. Fatty acid composition Saturated C12:0 C14:0 C16:0 C17:0 C18:0 C18:0 10-methyl C19:0 C20:0
AIC1e3
1.5 2.0 1.5
AIC2e15
26.8
11.6 3.9 9.2
AIC3e9
31.9
AIC4e13
AIC6e5
AIC6e11
AIC6e14
AIC9e14
11.1
23.7 12.7
4.8 4.4 6.1
2.4 2.0 1.3
4.9 13.7
6.4
6.1
1.7
3.5
44.5
24.9
9.2
11.1
15.6
Total Branched iso-C12:0 iso-C13:0 anteiso-C13:0 iso-C14:0 iso-C14:0 3OH iso-C15:0 anteiso-C15:1 anteiso-C15:0 iso-C16:0 anteiso-C16:0 iso-C17:0 iso-C 17:1 u10c anteiso-C17:0 anteiso-C17:1 u9c iso-C 18:0 2OH iso-C19:0 anteiso-C19:0 iso-C20:0 Summed feature 4a Summed feature 9a
5.0
Total Unsaturated * C13:1 C15:1 u5C C16:1 u9c C16:1 u11c C16:1 2OH C 17:1 u8c C18:1 u9c C18:1 2OH C18:03(u6c) (6,9,12) Summed feature 3a Summed feature 5a
92.5
36.0
24.7
47.5
AIC10e14
AIC11e14
0.5 6.3 21.0 0.7 0.5 23.75
10.1 3.6 13.2 5.9 4.8
5.5
11.1
5.7
35.2
2.1 2.2 52.8
41.9
24.2 4.4 1.5
5.9 1.0 2.4
1.5 2.8
20.0
3.7
48.3 2.3
22.6 3.3
5.5 1.4 4.6
4.4
16.4 0.1
2.2
4.2 1.9
6.6
4.8 18.6 4.1
1.4
4.8 3.2 5.8
6.5 1.9
4.9 8.8 66.3 8.2
1.4 2.4
0.2 3.0 2.9
3.2 2.0 2.2
0.4 5.3
2.3
3.0
0.2
3.9 3.4
1.6 0.7 0.3 0.9 1.0
59.3
1.1 3.9 5.9
0.3 2.0
8.7 53.7
0.3
24.8
42.7
26.5
69.1
92.0
44.0
6.4
8.2
2.8
2.6
0.2 0.6 0.4
3.2
3.2 2.4
0.1
2.5 22.8 5.9
6.8 0.9 1.6
2.5 13.1 62.2
10.5
1.2 3.4
11.4
6.9
6.0
1.5 12.4
26.1
24.0
AIC10e14 1.1 0.3
AIC11e14
9.2
Fatty acid composition Summed feature 6a Summed feature 7a Summed feature 8a
AIC1e3
Total
2.5
*
AIC 1e25
AIC 1e25
9.2
AIC2e15
AIC3e9
AIC4e13
11.8
3.6 13.5
12.7
74.1
27.6
42.6
AIC6e5
AIC6e11
AIC6e14
AIC9e14 3.9
17.9 29.1
6.0
2.8
19.3
42.6
44.1
Unsaturated includes C13:1, C15:1 u9c, C16:1 u9c, C16:1 u11c, C17:1 u8c, C18:1 u9c, C18:3 (6, 9, 12), summed feature 3, summed feature 6, summed feature 7 and summed feature 8. Iso- þ anteiso includes all the iso- and anteiso fatty acids indicated in the table. Saturated includes C12:0, C14:0, C16:0, C17:0, C18:0, C19:0 and C20:0. a Summed features represent groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 3-(C16:1 u7c/16:1 u6c); summed feature 4-(iso-C17:1 I/anteiso-C17:1 B); summed feature 5-(anteiso-C18:0/C18:2 u6,9c); summed feature 6-(C19:1 u11c/C19:1 u9c); summed feature 7-(C19:1 u7c/ C19:1 u6c); summed feature 8-(C18:1 u7c/C18:1 u6c); summed feature 9-(iso-C17:1 u9c/C16:0 10-methyl).
Supplementary Table 3 Fatty acid profiles of the 14 representative Gram-positive bacterial strains among the 25 bacterial groups isolated from the 5 different depths (AIC-2; AIC-3; AIC-5; AIC-10; AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica. Fatty acid composition Saturated C12:0 C12:0 3OH C14:0 C15:0 2OH C16:0 C17:0 2OH C17:0 10-methyl C18:0 C19:0 C20:0
Fatty acid composition iso-C17:0 iso-C17:0 3OH iso-C 17:1 u10c anteiso-C17:0 anteiso-C17:1 u9c iso-C18:1 H iso-C18:0 iso-C19:0 anteiso-C19:0 iso-C20:0 Summed Feature 2a Summed Feature 4a Summed Feature 9a Total
AIC 3e7
AIC 3e16
0.6 16.3
16.3
0.8
11.2
0.3
0.6
0.2
0.5
1.9
12.3
2 1
1.42 1.0 0.8
AIC 3e19 1.5 3.3 3.0 0.8 5.7 1.6
AIC 3e20
AIC 5e1
3.8 32.7
24.2
5.6
8.1 5.1 6.0
14.2 10.1 10.8
21.5
55.7
59.3
AIC 5e18
AIC 5e21
AIC 5e24
0.6
0.5
5.3
0.4
0.6
35.9
1.0
1.4
1.0 4.8
0.3 0.3
47.6
2.5
2.0
1.0
11.3 10.2 24.7
0.8 12.4
1.0 8.5
69.3 0.3 0.4
7.4
AIC 5e28
AIC 10e21
2.1
4.4
3.4
6.4
8.2
13.7
AIC3e7
AIC3e16
13.0
0.7
7.5
13.0
1.2 3.3
2.0 2.2
AIC 11e28
1.5
0.8
10.2
2.7
6.2
4.5
1.0
2.9
3.7 3.1 2.0
15.3
11.2
7.1
15.8
6.5
3.8
3.2
3.9
4.3
1.1
2.3 0.4
2.5
8.9
0.5
41.7
6.3
1.9
3.8
1.2 0.4
27.7 1.5 0.5
53.5 2.7
32.5 3.2
13.0
0.4 1.6
3.3
21.9
8.1
AIC3e19
4.8 2.0 2.5
AIC3e20
3.2 7.7
AIC5e1
6.3
8.5
AIC5e18
11.3 3.5
AIC5e21
AIC5e24
0.8
0.7
0.8 1.5 0.3
1.1 4.4
AIC10e21
AIC11e10
AIC11e11
19.2
3.0
9.9
1.8
15.9
6.2 13.6 35.7
1.5 5.0 4.3
49.0
AIC11e28
8.0 1.4 1.1
2.8
3.6 0.4 1.0
8.3
79.2
85.9
3.0 2.7
6.4 95.5
1.4
14.1
AIC5e28
1.2
3.7
83.6
1.0 41.1 9.5 1.6
1.9
11.4 0.4
AIC 11e11
0.9
1.0 AIC2e22
AIC 11e10
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
Total Branched iso-C12:0 anteiso-C12:0 iso-C13:0 anteiso-C13:0 iso-C14:0 iso-C14:0 3OH anteiso-C14:0 iso-C15:0 iso-C15:1 anteiso-C15:1 anteiso-C15:0 iso-C16:0 iso-C16:1 u11c anteiso-C16:0
AIC 2e22
13.9
36.1
22.9
28.5
79.6
79.5
81.0
27.8
8.8 79
(continued on next page)
80
Supplementary Table 3 (continued ) Fatty acid composition
AIC 2e22
Unsaturated * C12:1 3OH C15:1 u6c C15:1 u5c C16:1 u9c C16:1 u7c alcohol C16:1 u11c C 17:1 u6c C18:1 C18:1 u9c C18:1 u7c 11-methyl C18:3 u6,9,12c C20:2 u6,9C C20:1 u9c C20:1 u7c Summed Feature 3a Summed Feature 5a
AIC 3e16
AIC 3e19
AIC 3e20
AIC 5e1
AIC 5e18
AIC 5e21
AIC 5e24
AIC 5e28
AIC 10e21
AIC 11e10
AIC 11e11
6.1
2.3 53.9
AIC 11e28
1.1 2.5 1.5
1.9
6.2 0.3
1.2
1.1 0.4
7.0 3.5
1.4
4.0 14.7 0.6
9.8
5.5
0.5
1.5
8.1 1.0 1.0
0.3 0.2
8.0
2.0
0.4 AIC 2e22
Total
0
11.9
12.7 1.9
AIC 3e7
AIC 3e16
AIC 3e19
0.2
11.2
26.6
2.2
31.3
56.9
0.4 11.2 0.3
0.2
4.2 1.1
1.1 2.2
AIC 3e20 3.6
AIC 5e1
AIC 5e18 3.2 0.9
AIC 5e21 0.8 0.2
AIC 5e24
AIC 5e28 4.0
AIC 10e21
AIC 11e10
AIC 11e11
AIC 11e28 39.2 23.7
3.6
17.8
24.2
17.9
11.6
5.9
2.2
7.7
58.1
70.1
Unsaturated includes C13:1, C15:1 u9c, C16:1 u9c, C16:1 u11c, C17:1 u8c, C18:1 u9c, C18:3 (6, 9, 12), summed feature 3, summed feature 6, summed feature 7 and summed feature 8. Iso- þ anteiso includes all the iso- and anteiso fatty acids indicated in the table. Saturated includes C12:0, C14:0, C16:0, C17:0, C18:0, C19:0 and C20:0. a Summed features represent groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 2-(iso-C16:1 I/C14:0 3OH); summed feature 3-(C16:1 u7c/16:1 u6c); summed feature 4-(iso-C17:1 I/anteiso-C17:1 B); summed feature 5-(anteiso-C18:0/C18:2 u6,9c); summed feature 7-(C19:1 u7c/C19:1 u6c); summed feature 8-(C18:1 u7c/C18:1 u6c); summed feature 9-(iso-C17:1 u9c/C16:0 10methyl).
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
0.7
Fatty acid composition Summed feature 7a Summed feature 8a
*
AIC 3e7
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
81
Supplementary Table 4 Affiliation of the 138 bacterial strains, isolated from the 11 different depths (AIC-1 to AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica, to phyla, genera and species/subspecies based on analysis of complete 16S rRNA gene sequences. Phylum
Genus
Species/subspecies
AIC samples 1
Actinobacteria
Firmicutes
Arthrobacter Brevibacterium Rhodococcus
Bacillus Brevibacillus Paenibacillus
Sporosarcina Staphylococcus Bacteroidetes Proteobacteria
Flavobacterium Sphingobacterium Janthinobacterium Naxibacter Pseudomonas
Sphingomonas Stenotrophomonas
Arthrobacter rhombi Brevibacterium antiquum Rhodococcus cercidiphylli Rhodococcus fascians Rhodococcus qingshengii Bacillus psychrodurans Bacillus stratosphericus Brevibacillus centrosporus Brevibacillus panacihumi Paenibacillus humicus Paenibacillus peoriae Paenibacillus xylanexedens Sporosarcina globispora Staphylococcus sciuri subsp. sciuri Flavobacterium pectinovorum Sphingobacterium anhuiense Janthinobacterium lividum Naxibacter haematophilus Pseudomonas azotoformans Pseudomonas brenneri Pseudomonas poae Pseudomonas stutzeri Sphingomonas aerolata Sphingomonas faeni Stenotrophomonas rhizophila
2
1 2
Total
3
4
5 1 1 2 2 1
1
6
7
8
9
10
7 1
11
3
1 1 1
1 1 6 4
3
3
3 1 1 1 1 2
2 6
4
1
3 1
1
24
11
3 5
4
1 1 4
10
Acknowledgments We would like to thank the Council of Scientific and Industrial Research and NCAOR, Ministry of Earth Sciences, Government of India, for financial support to SS. TNRS acknowledges the CSIR, Government of India for the award of a Research Associateship. References Abyzov, S.S., Bobin, N.E., Koudryashov, B.B., 1982. Quantitative assessment of microorganisms in microbiological studies of Antarctic glaciers. Biol. Bull. Acad. Sci. 9, 558e564. Abyzov, S., Mitskevich, I., Poglazova, M., 1998. Microflora of the deep glacier horizons of Central Antarctica. Microbiology 67, 66e73. Abyzov, S.S., Mitskevich, I.N., Poglazova, M.N., Barkov, N.I., Lipenkov, V.Y., Bobin, N.E., Koudryashov, B.B., Pashkevich, V.M., Ivanov, M.V., 2001. Microflora in the basal strata at Antarctic ice core above the Vostok lake. Adv. Space Res. 28, 701e706. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. Mol. Biol. 215, 403e410. Anil Kumar, P., Srinivas, T.N.R., Madhu, S., Ruth Manorama, R., Shivaji, S., 2010. Indibacter alkaliphilus gen. nov., sp. nov., a novel alkaliphilic bacterium isolated from haloalkaline Lonar Lake, India. Int. J. Syst. Evol. Microbiol. 60, 721e726. Castello, J., Rogers, S., 2005. Life in Ancient Ice. Princeton University Press, Princeton, NJ, p. 300. Cheng, S.M., Foght, J.M., 2007. Cultivation-independent and -dependent characterization of bacteria resident beneath John Evans glacier. FEMS Microbiol. Ecol. 59, 318e330.
13
10
1 3 2
7 12
10
2 11
25
12
16
10
9
Number
%
1 1 2 13 6 1 1 1 1 6 10 3 1 1
0.7 0.7 1.4 9.4 4.3 0.7 0.7 0.7 0.7 4.3 7.2 2.2 0.7 0.7
1 1 4 7 54 1 2 2 3 6 9 138
0.7 0.7 2.9 5.1 39.1 0.7 1.4 1.4 2.2 4.3 6.5 100
Chintalapati, S., Kiran, M.D., Shivaji, S., 2004. Role of membrane lipid fatty acids in cold adaptation. Cell. Mol. Biol. 50, 631e642. Christner, B.C., Mosley-Thompson, E., Thompson, L.G., Reeve, J.N., 2001. Isolation of bacteria and 16S rDNAs from Lake Vostok accretion ice. Environ. Microbiol. 3, 570e577. Christner, B.C., Thompson, E.M., Thompson, L.G., Zagorodnov, V., Sandman, K., Reeve, J.N., 2002. Isolation and identification of bacteria from ancient and modern ice core archives. In: Casassa, G., Sepu´lveda, F.V., Sinclair, R. (Eds.), The Patagonian Ice Fields. A Unique Natural Laboratory for Environmental and Climate Change Studies. Kluwer Academic/Plenum Publishers, New York. Feller, G., Gerday, C., 2003. Psychrophilic enzymes: hot topics in cold adaptation. Nat. Rev. Microbiol. 1, 200e208. Foreman, C.M., Dieser, M., Greenwood, M., Cory, R.M., Laybourn-Parry, J., Lisle, J.T., Jaros, C., Miller, P.L., Chin, Y.P., McKnight, D.M., 2011. When a habitat freezes solid: microorganisms over-winter within the ice column of a coastal Antarctic lake. FEMS Microbiol. Ecol. 76, 401e412. Johnson, S.S., Hebsgaard, M.B., Christensen, T.R., Mastepanov, M., Nielsen, R., Munch, K., Brand, T., Gilbert, T., Zuber, M.T., Bunce, M., Ronn, R., Gilichinsky, D., Froese, D., Willerslev, E., 2007. Ancient bacteria show evidence of DNA repair. Proc. Natl. Acad. Sci. 104, 14401e14405. Karl, D.M., Bird, D.F., Bjorkman, K., Houlihan, T., Shackelford, R., Tupas, L., 1999. Microorganisms in the accreted ice of Lake Vostok, Antarctica. Science 286, 2144e2147. Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic Acid Techniques in Bacterial Systematics. Wiley, Chichester, pp. 115e175. Miteva, V.I., Sheridan, P.P., Brenchley, J.E., 2004. Phylogenetic and physiological diversity of microorganisms isolated from a deep Greenland glacier ice core. Appl. Environ. Microbiol. 70, 202e213. Moore, L.R., Rocap, G., Chisholm, S.W., 1998. Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature 393, 464e467.
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Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones. Extremophiles 15, 1e22. Sleight, S.C., Wigginton, N.S., Lenski, R.E., 2006. Increased susceptibility to repeated freeze-thaw cycles in Escherichia coli following long-term evolution in a benign environment. BMC Evol. Biol. 6, 104. Srinivas, T.N.R., Nageswara Rao, S.S.S., Vishnu Vardhan Reddy, P., Pratibha, M.S., Sailaja, B., Kavya, B., Hara Kishore, K., Begum, Z., Singh, S.M., Shivaji, S., 2009. Bacterial diversity and bioprospecting for cold-active lipases, amylases and proteases, from culturable bacteria of ˚ lesund, Svalbard. Arctic. Curr. Microbiol. 59, Kongsfjorden and Ny-A 537e547. Steven, B., Briggs, G., McKay, C.P., Pollard, W.H., Greer, C.W., Whyte, L.G., 2007. Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and cultureindependent methods. FEMS Microbiol. Ecol. 59, 513e523. Thamban, M., Chaturvedi, A., Rajakumar, A., Naik, S.S., D’Souza, W., Singh, A., Rajan, S., Ravindra, R., 2006. Aerosol perturbations related to volcanic eruptions during the past few centuries as recorded in an ice core from the Central Dronning Maud Land, Antarctica. Curr. Sci. 91, 1200e1207. Thompson, L.G., Yao, T., Mosley-Thompson, E., Davis, M.E., Henderson, K.A., Lin, P.N., 2000. A high-resolution millennial record of the South Asian monsoon from Himalayan ice cores. Science 289, 1916e1919. Walker, V.K., Palmer, G.R., Voordouw, G., 2006. Freeze-thaw tolerance and clues to the winter survival of a soil community. Appl. Environ. Microbiol. 72, 1784e1792. Xiang, S., Yao, T., An, L., Xu, B., Wang, J., 2005. 16S rRNA sequences and differences in bacteria isolated from the Muztag Ata glacier at increasing depths. Appl. Environ. Microbiol. 71, 4619e4627. Zhang, X., Yao, T., Ma, X., 2002. Microorganism in a high Altitude Glacier Ice in Tibet, China. Folia. Microbiologia 47, 241e245. Zhang, X., Ma, X., Yao, T., Zhang, G., 2003. Diversity of 16S rDNA and environmental factor influencing microorganisms in Malan ice core. Chin. Sci. Bull. 48, 1146e1151. Zhang, X., Yao, T., An, L., Tian, L., Xu, S., 2006a. Study on vertical profile of bacterial DNA Structure in Puruogangri Ice Core by denaturing gradient gel electrophoresis. Ann. Glaciol 43, 160e166. Zhang, S., Hou, S., Ma, X., Qin, D., Chen, T., 2006b. Culturable bacteria in Himalayan ice in response to atmospheric circulation. Biogeosci. Diss. 3, 765e778. Zhang, X.F., Yao, T.D., Tian, L.D., Xu, S.J., An, L.Z., 2008. Phylogenetic and physiological diversity of bacteria isolated from Puruogangri ice core. Microb. Ecol. 55, 476e488.
Antarctic ice core samples: culturable bacterial diversity Sisinthy Shivaji a,*, Zareena Begum a, Singireesu Soma Shiva Nageswara Rao a, Puram V. Vishnu Vardhan Reddy a, Poorna Manasa a, Buddi Sailaja a, Mambatta S. Prathiba a, Meloth Thamban b, Kottekkatu P. Krishnan b, Shiv M. Singh b, Tanuku N.R. Srinivas a a
b
Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India National Center for Antarctic and Ocean Research, Headland Sada, Vasco da Gama, Goa, India Received 24 October 2011; accepted 4 September 2012 Available online 4 October 2012
Abstract Culturable bacterial abundance at 11 different depths of a 50.26 m ice core from the Tallaksenvarden Nunatak, Antarctica, varied from 0.02 to 5.8 103 CFU ml1 of the melt water. A total of 138 bacterial strains were recovered from the 11 different depths of the ice core. Based on 16S rRNA gene sequence analyses, the 138 isolates could be categorized into 25 phylotypes belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. All isolates had 16S rRNA sequences similar to previously determined sequences (97.2e100%). No correlation was observed in the distribution of the isolates at the various depths either at the phylum, genus or species level. The 25 phylotypes varied in growth temperature range, tolerance to NaCl, growth pH range and ability to produce eight different extracellular enzymes at either 4 or 18 C. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a significant proportion of the total fatty acid composition. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: 16S rRNA gene; Actinobacteria; Bacteroidetes; Firmicutes; Proteobacteria; Phenotypic characteristics; Extracellular enzymatic activities; Fatty acid profiles
1. Introduction Ice cores offer a unique opportunity to correlate microbial diversity with age and prevailing chemical conditions in ice cores (Castello and Rogers, 2005). Studies on ice cores from the Malan glacier, the Muztag Ata glacier and the Puruogangri glacier (Zhang et al., 2003; Xiang et al., 2005) established that bacterial abundance positively correlated with dust within the ice core (Zhang et al., 2006a). Furthermore, the phylogenetic diversity of bacteria in the ice cores revealed a high abundance
* Corresponding author. Tel.: þ91 40 27192504; fax: þ91 40 27160311. E-mail addresses: [email protected] (S. Shivaji), [email protected] (Z. Begum), [email protected] (S.S. Shiva Nageswara Rao), [email protected] (P.V. Vishnu Vardhan Reddy), [email protected] (P. Manasa), [email protected] (B. Sailaja), [email protected] (M.S. Prathiba), [email protected] (M. Thamban), [email protected] (K.P. Krishnan), [email protected] (S.M. Singh), [email protected] (T.N.R. Srinivas).
of members of the phyla Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes in Tibetan ice cores, the Puruogangri Ice core,the Muztag Ata glacier ice core and the Deep Greenland glacier ice core (Miteva et al., 2004; Xiang et al., 2005; Zhang et al., 2002, 2003, 2008). In some ice cores, as in the Greenland glacier, in addition to aerobic bacteria, anaerobic bacteria related to Thermus, Bacteroides, Eubacterium and Clostridium groups were also found (Miteva et al., 2004; Moore et al., 1998). Studies on species diversity, genetics and physiological differences in ice core isolates is crucial, since bacteria with nearly or fully matching 16S rRNA gene sequences exhibit distinct physiological capabilities (Moore et al., 1998). Despite the above studies, very few studies have been carried out on ice cores from Antarctica (Abyzov et al., 2001; Christner et al., 2001, 2002; Foreman et al., 2011; Karl et al., 1999). This is all the more important, since psychrophilic bacteria at the species level need to be documented and studied to a greater extent so as to be able to exploit their
0923-2508/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.resmic.2012.09.001
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
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potential for biotechnological application (Feller and Gerday, 2003). Some psychrophilic bacteria are able to produce omega-3 polyunsaturated fatty acids which are of dietary significance (Nichols et al., 1995). Psychrophilic bacteria are also potential sources of novel pigments (they can be used as food additives) and cold-active enzymes for industrial application (Feller and Gerday, 2003). The goal of the present study was to establish bacterial abundance and viable bacterial diversity in an ice core sample collected from the continental ice sheet at central Dronning Maud Land, Antarctica. In addition, these bacteria were also used for bioprospecting for fatty acids and cold-active enzymes.
Research, Goa, India, in a cold room facility (20 C). Prior to chemical and microbiological studies, the surface of the ice core was scraped with a sterile microtome blade and then a slice of ice from a particular depth was cut and separated using a sterile band-saw machine (Table 1). Only the inner unexposed core of the sample was used for microbiological analysis. Utmost care was taken to minimize contamination during sectioning and subsequent analysis. The core samples were melted in a class 100 clean room before analysis. The age of the ice core was estimated according to the method employed earlier (Thamban et al., 2006).
2. Materials and methods
The melt water from the ice core sample was filtered through a 0.2 mm filter. Subsequently, the filter was placed on Antarctic bacterial medium (ABM) plates [peptone (0.5%, w/v), yeast extract (0.2%, w/v) and agar (2%, w/v)] and incubated at 10 C for 15 days. Different morphotypes that appeared were purified and maintained on ABM plates.
2.1. Sampling site and sampling method An ice core (IND-22/B4) of 50.26 m was recovered from the Tallaksenvarden nunatak located in central Dronning Maud Land (70 51.30 S and 11 32.20 E, elevation 680 m), Antarctica (Fig. 1), during the 22nd Indian Scientific Expedition to Antarctica. The annual mean temperature and snow accumulation reported at this site were w20 C and w170 kg m2 a1 respectively. Ice core drilling was carried out during the austral summer (FebruaryeMarch) of 2003 using an electromechanical drilling system (diameter w10 cm, type D-2; GeoTecs Ltd., Japan) and the retrieved ice core samples were labeled, packed in polythene containers and shipped to India in a deep-freezer at 20 C. Subsequently, the samples were archived in polypropylene insulated containers at the National Center for Antarctic and Ocean
2.2. Isolation and culturing of bacteria
2.3. Characterization of bacterial strains Colony characteristics were observed with the help of a magnifying lens. Growth at different temperatures, pH and salt concentrations were carried out on ABM plates. Extracellular enzymatic activities of amylase, lipase and proteases were checked by streaking the culture on ABM plates supplemented with 0.2% soluble starch or 1% Tween-60 along with 0.01% CaCl2 or 0.3% casein, respectively, incubated at 4 C and 18 C for 5e10 days. Gelatinase activity was checked by inoculating the culture in ABM broth supplemented with 10% gelatin and incubating the plates at 4 C and 18 C for 5e10 days. DNase, esculinase and urease activities were checked by streaking the culture on DNase test agar (M1041, HIMEDIA, Hyderabad, India), esculin agar (M1386, HIMEDIA, Hyderabad, India) or urea agar base (M112, HIMEDIA, Hyderabad, India) and by subsequently incubating the plates at 4 C and 18 C for 5e10 days. Catalase activity was tested by adding hydrogen peroxide to the culture taken on a slide. Fatty acid methyl esters were prepared and analyzed as previously described (Shivaji et al., 2007) using cultures grown at 18 C. 2.4. Tolerance to freezing and freeze-thaw
Fig. 1. Ice core sample collection point (IND-22/B4) in the Dronning Maud Land, Antarctica.
Tolerance of the bacteria to freezing and freeze-thaw was done by previously described methods (Sleight et al., 2006; Walker et al., 2006). Six cultures were selected based on their growth temperature range [AIC 11.11 (4e18 C), AIC 5.21 and AIC 5.24 (4e30 C) and AIC 3.7, AIC 5.1 and AIC 11.14 (4e37 C)] and grown in ABM broth for 5 days till they reached stationary phase. Then, 1 ml of the culture was taken and centrifuged at 8000 rpm at room temperature for 10 min. The cell pellet was suspended in distilled water mixed properly and the tube was placed in a 20 C freezer. Simultaneously, 18 tubes were prepared and 15 of them were frozen at
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Table 1 Bacterial abundance and physical characteristics at the 11 different depths (AIC-1 to AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica. CFU (103)/ml
Number of morpho-types
Strain numbers
4.8 0.3 4.0 0.9
10 13
17.72
3.2 1.0
10
1859
19.06
3.1 0.4
12
20.35e20.45
1814
19.22
3.3 0.4
10
6.4
25.11e25.21
1772
17.73
4.5 1.0
11
AIC7
6.5
30.30e30.4
1723
15.47
5.8 0.6
25
8
AIC8
6.4
34.88e34.98
1680
17.90
4.8 0.7
12
9
AIC9
6.4
40.08e40.18
1631
17.76
3.8 1.0
16
10
AIC10
6.4
44.88e44.98
1586
18.11
1.7 0.3
10
11
AIC11
6.4
50.16e50.26
1535
19.31
0.02 0.001
9
AIC1e1 to 1e6; 1e8; 1e25; 1e26; 1e29 AIC2e1; 2e5; 2e7 to 2e11; 2e13 to 2e15; 2e22; 2e27; 2e28 AIC3e7 to 3e10; 3e12B; 3e16; 3e19 to 3e22 AIC4e1; 4e2; 4e5 to 4e8; 4e10 to 4e14; 4e18 AIC5e1; 5e7 to 5e10; 5e18; 5e21; 5e24; 5e25; 5e28 AIC6e1; 6e4; 6e5; 6e11A; 6e11B; 6e14; 6e24; 6e26; 6e27; 6e29; 6e30 AIC7e2; 7e5 to 7e8; 7e10 to 7e18; 7e20; 7e21; 7e23 to 7e31 AIC8e2 to 8e6; 8e9; 8e14; 8e26 to 8e28; 8e31; 8e32 AIC9e1 to 9e6; 9e8; 9e10; 9e12 to 9e17; 9e20; 9e26 AIC10e9; 10e10; 10e13 to 10e16; 10e21; 10e25; 10e30; 10e31 AIC11e10; 11e11; 11e14; 11e19; 11e20; 11e23; 11e24; 11e28; 11e30
Sl.no.
Sample ID
pH
1 2
AIC1 AIC2
7 6.8
3
AIC3
4
Depth (m)
Age (AD)
Ice core surface temperature ( C)
0.25e0.35 5.06e5.16
1993 1940
18.85 21.57
6.7
10.36e10.46
1892
AIC4
6.5
15.26e15.36
5
AIC5
6.4
6
AIC6
7
20 C. Subsequently, at each time point (5, 9, 12, 14 and 15 days of freezing), 3 tubes were removed from the freezer, thawed for 1.5 h at 22 C and then 100 ml of the thawed culture was serially diluted in 900 ml of 0.9% saline. The diluted culture (100 ml of the suspension) was plated on TSA plates and incubated at 18 C for 3 days. Colony counts were recorded in triplicate, the average calculated and used for determining the percentage of cell survival, considering the cell count on day 0 as 100%. The remaining 3 tubes that were not frozen served as the zero time point control and were plated as above on day 0. Tolerance of the six cultures to freeze-thaw cycles was also tested. In these experiments, one ml of the culture in triplicate was prepared as above and frozen at 20 C. At the respective cycle number (0, 3, 7, 9, 11, 13 and 20), the cultures in triplicate were thawed for 1 h and then 100 ml of the thawed culture was serially diluted, plated and processed for viable counts as above. Each freeze-thaw cycle consisted of 1 h freezing at 20 C and thawing for 1.5 h at 22 C.
phylogenetic trees were constructed using the maximum likelihood (ML) method (Anil Kumar et al., 2010).
2.5. PCR amplification of 16S rRNA gene sequencing and phylogenetic analysis
3.2. Bacterial count
The 16S rRNA gene was amplified using pA (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and pH* (50 -AAG GAG GTG ATC CAG CCG CA-30 ) as previously described (Shivaji et al., 2006) and the resulting amplicons were sequenced using different internal primers (Lane, 1991). The 16S rRNA gene sequences were then subjected to BLAST sequence similarity search (Altschul et al., 1990) to identify the nearest taxa. Subsequently, the sequences were aligned with the sequences from the nearest taxa using the CLUSTAL_X program and
2.6. Nucleotide sequence accession numbers All 16S rRNA gene sequences of the strains were deposited in GenBank with accession numbers JF970575 to JF970601. 3. Results and discussion 3.1. Characteristics of ice core samples collected from Dronning Maud Land, Antarctica The pH in the ice core sample was neutral at the surface, but at all other depths up to 50 m, the pH was slightly acidic. Further, the estimated deposition date of the ice core ranged from 1993 AD for the surface 0.25 m layer to 1535 AD at a depth of 50.26 m. The ice core surface temperature varied at different depths of the ice core (Table 1).
The culturable bacterial count (colony-forming units, CFUs) in the Antarctic ice core varied from 0.02 to 5.8 103 CFU ml1 of the ice core melt water (Table 1). The minimum number was recorded at a depth of 50.16 m (Table 1). This is similar to that observed for Puruogangri ice core melt water, where CFU fluctuated from 0 to 760 CFU ml1 (Zhang et al., 2008). Earlier studies also indicated that the estimates of microbes in glacial ice differed widely with the geographic location, and the number varied from less than one viable cell ml1 in polar ice (Abyzov et al., 1982) to
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6 107 cells ml1 in a Greenland ice core (Sheridan et al., 2003). Further, the CFU ml1 of the ice core melt water did not reveal any appreciable tendency to change with increasing depth, thus implying that the microorganisms were episodically deposited in the glacier (Zhang et al., 2008). 3.3. Viable bacteria A total of 138 bacterial strains were recovered from the 11 different depths of the ice core (Tables 1 and 2). These 138 strains, based on similarity criteria of 97% at the 16S rRNA gene sequence level, could be categorized into 25 phylotypes (Table 2). Each group represented a different taxon (Table 2),
73
and since 97% sequence similarity was used as the cut-off, the groups varied with the nearest phylogenetic neighbor at the species level. Based on Gram staining, 11 groups were Gram-negative and 16 were Gram-positive. Out of the 25 phylotypes, 11 were common to two or more depths of the ice core and the most common phylotype was Pseudomonas azotoformans, which was shared by 7 out of the 11 depths of the ice core (Supplementary Table 4). 3.4. Characteristics of the isolated strains A representative from each of the 25 phylotypes was chosen to study some of their phenotypic (NaCl tolerance,
Table 2 Identification of the 138 bacterial strains isolated from the 11 different depths (AIC-1 to AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica based on BLAST analysis of the 16S rRNAa gene sequences. Sl.No.
Strain number
Nearest phylogenetic neighbor and 16S rRNA gene sequence similarity (%)
AIC5e18 AIC5e28 AIC5e24, 5e25 AIC1e8, 5e9, 5e10, 9e1 to 6, 9e8, 11e23, 11e28, 11e30 AIC8e9, 1e26, 1e29, 2e11, 5e21, 6e26
Arthrobacter rhombi F.98.3HR.69T Y15885, (99.2) Brevibacterium antiquum VKM Ac-2118T AY243344, (98.5) Rhodococcus cercidiphylli YIM 65003T EU325542, (99.1e99.4) Rhodococcus fascians DSM 20669T X79186, (99.4e99.9)
Bacillus psychrodurans DSM 11713T AJ277984, (99.4) Bacillus stratosphericus 41KF2aT AJ831841, (99.9) Brevibacillus centrosporus DSM 8445T AB112719, (99.8) Brevibacillus panacihumi DCY35T EU383033, (97.9) Paenibacillus humicus PC-147T AM411528, (98.7e99.0) Paenibacillus peoriae DSM 8320T AJ320494, (99.1e99.7)
12 13 14
AIC11e10 AIC10e21 AIC3e19 AIC3e7 AIC2e1, 2e5, 2e7, 2e22, 2e27, 2e28 AIC4e2, 4e5, 4e6, 4e7, 5e1, 5e7, 5e8, 11e19, 11e20, 11e24 AIC3e20, 3e21, 3e22 AIC11e11 AIC3e16
Paenibacillus xylanexedens B22aT EU558281, (99.0e99.4) Sporosarcina globispora DSM 4T X68415, (99.6) Staphylococcus sciuri subsp. sciuri DSM 20345T AJ421446, (99.6)
Bacteroidetes 15 16
AIC6e11A AIC6e5
Flavobacterium pectinovorum DSM 6368T AM230490, (98.2) Sphingobacterium anhuiense CW186T EU364817, (99.9)
AIC6e11B, 2e14, 2e15, 6e1 AIC10e25, 10e30, 10e31, 10e9, 9e26, 9e14, 9e10 AIC1e1, 1e2, 1e4 to 1e6, 1e25, 2e8 to 2e10, 2e13, 4e10, 6e24, 6e27, 6e29, 7e2, 7e5 to 7e8, 7e10, 7e11, 7e13 to 7e18, 7e20, 7e21, 7e23 to 7e31, 8e2 to 8e6, 8e14, 8e26 to 8e28, 8e31, 8e32, 9e12, 9e15 to 9e17, 9e20 AIC6e14 AIC7e12, 1e3 AIC11e14, 9e13 AIC10e10, 10e14, 10e16 AIC3e8, 3e9, 3e10, 3e12B, 10e13, 10e15 AIC4e1, 4e8, 4e11, 4e12, 4e13, 4e14, 4e18, 6e4, 6e30
Janthinobacterium lividum DSM 1522T Y08846, (99.6e99.7) Naxibacter haematophilus CCUG 38318T AM774589, (98.5e98.9)
Actinobacteria 1 2 3 4 5 Firmicutes 6 7 8 9 10 11
Proteobacteria 17 18 19
20 21 22 23 24 25
Rhodococcus qingshengii djl-6T DQ090961, (99.9e100.0)
Pseudomonas azotoformans IAM1603T D84009, (97.5e99.9)
Pseudomonas brenneri CFML 97-391T AF268968, (99.5) Pseudomonas poae DSM 14936T AJ492829, (99.3e99.9) Pseudomonas stutzeri CCUG 11256T U26262, (100.0) Sphingomonas aerolata NW12T AJ429240, (99.2) Sphingomonas faeni MA-olkiT AJ429239, (97.2e99.4) Stenotrophomonas rhizophila e-p10T AJ293463, (97.5e100.0)
a The 16S rRNA gene sequences of all 138 strains were obtained and subjected to BLAST and phylogenetic analyses. Based on phylogenetic analyses, the 138 strains were categorized into 25 groups and representatives of each group (indicated in bold) were submitted to EMBL (JF970575 to JF970601).
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temperature and pH range), biochemical (amylase, caseinage, catalase, DNase, esculinase, gelatinase, lipase and urease activities) and chemical (fatty acid composition) characteristics (Supplementary Tables 1e3). The 25 representative strains were either psychrophilic or psychrotolerant. The psychrophilic strains were able to grow between 4 and 18 C (5 strains), whereas the psychrotolerant strains were capable of growth at between 4 and 30 C (10 strains) or 4 and 37 C (10 strains) (Supplementary Table 1). Psychrotolerant bacteria capable of growing between 4 and 37 C were reported earlier (Srinivas et al., 2009; Reddy et al., 2009). The present observation that psychrotolerant bacteria (80.0%) are predominant, followed by the psychrophiles (20.0%), is in accordance with the earlier study of Xiang et al. (2005), who observed that bacteria isolated from the Muztag Ata glacier at increasing depths were predominantly psychrotolerant (82%), followed by psychrophiles (11%) and then mesophiles (7%). The Muztag Ata glacier ice isolates and the current isolates had temperature growth profiles consistent with adaptation to growth under cold growth conditions, and most likely were transferred by wind or snow from local ecosystems onto the glacier’s surface (Xiang et al., 2005). All strains were able to grow without NaCl in the medium, and tolerance to NaCl varied from 0.5 to 2 M (Supplementary Table 1). One of the most interesting results is that a high percentage of the isolates could grow in a very narrow pH range, with strains either growing only between pH 6e8, 6e9 and 7e9 (Supplementary Table 1). Strains having a narrow pH range for growth had been reported earlier (Srinivas et al., 2009; Reddy et al., 2009). 3.5. Tolerance of bacterial strains to freezing and freeze-thaw cycles Six bacterial strains were selected for the experiment based on their growth temperature range [AIC-11.11 (4e18 C), AIC-5.21 and AIC-5.24 (4e30 C) and AIC-3.7, AIC-5.1 and AIC-11.14 (4e37 C)] which varied from 4 to 18 to 4e30 and 4e37 C. For up to 5 days at 20 C, none of the cultures showed any appreciable decrease in survival, but by day 9 survival decreased and it was most pronounced in AIC-5.21, AIC-3.7, AIC-5.1 and AIC-11.14 (Fig. 3a). Subsequently, by day 15, survival of all cultures decreased to 10%. Culture AIC-11.11 Sporosarcina globispora, with a growth temperature range of 4e18 C, appeared to be the most tolerant to freezing (Fig. 3a) compared to the other cultures. Compared to continuous freezing, the above six cultures showed more resistance to freeze thaw cycles. Strains AIC3e7, AIC5e21, AIC11e14 and AIC11e11 showed >10% survival even after 20 freeze-thaw cycles. But AIC5e1 and AIC5e24 showed drastic decreases in growth after cycle 3 (Fig. 3b). This ability to resist freeze-thaw cycles is an adaptive feature required for survival in ice cores. However, the present study did not find any correlation between growth temperature range and percent survival after freezing or freeze thaw conditions.
The fatty acid profile data also support the hypothesis that these isolates adapt to a cold environment by possessing more branched and unsaturated fatty acids (analyzed by growing the cultures at 18 C) (Supplementary Tables 2 and 3). 3.6. Taxonomic analysis of the 25 representative strains The phylogenetic trees constructed to determine the affiliation of the representative strains of the 25 groups are shown in Fig. 2a and b. Among the 11 Gram-negative groups, 9 belonged to the phylum Proteobacteria and two to the phylum Bacteroidetes (Table 2 and Supplementary Table 4). The nearest phylogenetic neighbors of 9 of the 11 Gram-negative representative strains had been earlier isolated from cold habitats and were reported to be either psychrophilic or psychrotolerant, and include Janthinobacterium lividum (Reddy et al., 2009), P. azotoformans (Srinivas et al., 2009), Pseudomonas brenneri and Pseudomonas stutzeri (Srinivas et al., 2009), Pseudomonas poae, Sphingobacterium anhuiense, Sphingomonas aerolata, Sphingomonas faeni and Stenotrophomonas rhizophila. Only two strains, AIC6e11 and AIC9e14, related to Flavobacterium pectinovorum and Naxibacter haematophilus, respectively, had not been reported earlier from any cold habitat. Among the 16 Gram-positive groups, 5 were affiliated with the phylum Actinobacteria and the nearest phylogenetic neighbors were Arthrobacter rhombi (Osorio et al., 1999), Brevibacterium antiquum, Rhodococcus cercidiphylli, Rhodococcus fascians and Rhodococcus qingshengii (Srinivas et al., 2009) (Table 2 and Supplementary Table 4). Psychrophilic and psychrotolerant strains of the above nearest phylogenetic neighbors were earlier isolated from cold habitats like Greenland, permafrost samples and the Arctic. The remaining 11 Gram-positive groups were affiliated with the phylum Firmicutes and represented by genera/species of Bacillus, Brevibacillus, Lysinibacillus fusiformis, Paenibacillus, S. globispora and Staphylococcus sciuri (Table 2 and Supplementary Table 4). Only a few of the phylogenetic neighbors of the 11 groups affiliated with Firmicutes were isolated from cold habitats like the Alaskan tundra and the stratosphere, and they include Bacillus stratosphericus (Shivaji et al., 2006) and Paenibacillus xylanexedens. Four other psychrotolerant strains, namely Bacillus psychrodurans, Brevibacillus centrosporus, Paenibacillus peoriae and S. globispora, have not been reported from any cold habitat. Thus, the results indicate that most of the 25 representative strains are affiliated with species/genera from cold habitats except for those representative strains affiliated with the genera Lysinibacillus, Staphylococcus and Naxibacter. The occurrence of related phylotypes in geographically diverse cold environments suggests that they are tolerant to a cold environment and possess similar strategies to survive freezing and remain active at low temperatures (Abyzov et al., 1998). The ability of psychrophilic and psychrotolerant bacteria to adapt to cold conditions may be ascribed to the occurrence of pigments or polyunsaturated fatty acids, and with enzymes that are active
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82 Fig. 2. Phylogenetic trees based on 16S rRNA gene sequences showing the relationship of Gram-positive (a) and Gram-negative (b) strains obtained from the Antarctic ice core sample collected from the continental ice sheet at central Dronning Maud Land, Antarctica, with their nearest phylogenetic relatives. Phylogenetic trees were constructed by the by maximum likelihood method. Numbers at nodes are bootstrap values (50). Aquifex pyrophilus Kol5aT (M83548) was used as an outgroup. The bar represents 0.02 substitutions per alignment position in (a) and (b).
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S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
Fig. 3. Tolerance of bacteria to freezing (a) and freeze-thaw cycles (b). (a) Survival of 6 bacterial taxa up to a period of 15 days at 20 C was monitored. (b) Survival of the same 6 bacterial taxa to 20 cycles of freeze-thaw was monitored. Each freeze-thaw cycle consisted of 1 h freezing at 20 C and thawing at 22 C for 1.5 h. The bacterial taxa studied were (O) ¼ AIC 5:1; (,) ¼ AIC 3:7; (D) ¼ AIC 11:11; (C) ¼ AIC 5:24; (-) ¼ AIC 5:21; (:) ¼ AIC 11:14.
at low temperature (Chintalapati et al., 2004; Shivaji et al., 2007). Most of the isolates (79%) were found to be non-sporeformers and only 29 out of 138 isolates produced spores and belonged to the genera Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus and Sporosarcina. This is in accordance with the earlier observations that non-spore-forming bacteria are more abundant in glacial ice (Shafaat and Ponce, 2006). The abundance of non-spore formers in ice has been attributed to their ability to survive for extended periods of time in cold conditions because their DNA degrades far more slowly, and DNA repair is probably more efficient than that in spore formers (Johnson et al., 2007). 3.7. Vertical distribution of the isolates in the ice core The 138 isolates from the 11 depths of the ice core belonged to four different phyla: Actinobacteria (high G þ C), Firmicutes (low G þ C), Bacteroidetes and Proteobacteria (Table 2). Thus, current data support earlier data showing that members of the phyla Proteobacteria, Bacteroidetes and Actinobacteria are common in most cold habitats (Cheng and Foght, 2007; Neufeld et al., 2004; Shivaji et al., 2011; Steven et al., 2007; Srinivas et al., 2009; Reddy et al., 2009). In the
present study, the most frequently isolated bacteria belonged to the phylum Proteobacteria (61.5%) and were detected at all depths except in AIC-5 (20.35 m). The most predominant taxon was P. azotoformans, which was isolated from 7 depths of the ice core (Table 2 and Supplementary Table 4). Isolates affiliated with the phylum Firmicutes were the next most abundant (21%); they exhibited discontinuous distribution along the depth of the ice core and were not obtained on the surface or at depths corresponding to AIC-6 to AIC-9 (25.11e40.08 m respectively) (Table 2 and Supplementary Table 4). The dominant taxon among Firmicutes was Paenibacillus peoriae. Isolates affiliated with the phylum Actinobacteria constituted 16.1% of the total isolates and were represented by five taxon, all present in AIC-5 but absent in AIC-3, AIC-4, AIC-7 and AIC-10 (Table 2 and Supplementary Table 4). The phylum Bacteroidetes was the least represented (1.4%) and was present only in AIC-6 (25.11 m) (Table 2 and Supplementary Table 4). AIC-6 showed maximum diversity represented by 7 different species and AIC-7 and AIC-8 showed minimal diversity represented by only two species. Only P. azotoformans and Rhodococcus quingshengii were common to 5 depths of the ice core. The results indicate that distribution of taxa did not exhibit a specific pattern of distribution along the length of the ice core. For instance, Actinobacteria and Proteobacteria were present in AIC-1, AIC-8 and AIC-9, Actinobacteria, Firmicutes and Proteobacteria in AIC-2 and AIC-11, Firmicutes and Proteobacteria in AIC-3, AIC-4 and AIC-10, Actinobacteria and Firmicutes in AIC-5, Actinobacteria, Bacteroidetes and Proteobacteria in AIC-6 and only Proteobacteria in AIC-7 (Table 2 and Supplementary Table 4). Similar differential distribution of high G þ C, low G þ C, Bacteroidetes and Proteobacteria have been observed in ice cores from the Muztag Ata glacier (Xiang et al., 2005) and in the Puruogangri ice core (Zhang et al., 2008). Xiang et al. (2005) attributed the differential distribution to differences in bacteria deposited serendipitously on the glacier’s surface by wind and snowfall, and nutrient availability within the ice (Xiang et al., 2005). Proteobacteria, high G þ C, low G þ C and Bacteroidetes group bacteria were earlier also isolated from a Vostok (Antarctica) ice core (Christner et al., 2001) and the Siple glacier, Antarctica (Christner et al., 2001). 3.8. Extracelluar 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 25 representative strains showed activities of one or more enzymes (amylase, caseinase, catalase, DNase, esculinase, gelatinase, lipase or urease) either at 4 C and (or) 18 C (Supplementary Table 1). Only 3 strains (AIC2e15, AIC3e20 and AIC4e13) showed 5 out of 8 different enzymatic activities (Supplementary Table 1). Furthermore, 19 of the 25 representative strains produced extracellular enzymes at 4 C (Supplementary Table 1).
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
3.9. Fatty acid profiles In psychrophilic and psychrotolerant bacteria (all phylotypes), branched fatty acids varied in composition from a minimum of 0.3%, as in AIC2e15, to a maximum of 92.5% as in AIC1e3 (Supplementary Tables 2 and 3). In AIC2e15, the low % of branched fatty acids was compensated for by a high proportion of unsaturated fatty acids (74.1%) (Supplementary Table 2). The saturated fatty acid composition in all isolates varied in composition from a minimum of 2.0%, as in AIC5e24, to a maximum of 59.3, as in AIC5e1 (Supplementary Tables 2 and 3). These results are in accordance with earlier studies which indicated that unsaturated and branched (including the iso- and anteiso-) fatty acids are common in psychrophilic and psychrotolerant bacteria (Chintalapati et al., 2004) and play an important role in maintaining functional membrane fluidity crucial for continued survival at low temperatures (Nishida and Murata, 1996). Thus, it appears that psychrophilic and psychrotolerant bacteria from ice cores adapt to the cold environment by preferentially synthesizing unsaturated and branched fatty acids. 3.10. Conclusions Bacterial abundance in an ice core sample collected from the continental ice sheet at central Dronning Maud Land,
77
Antarctica, was observed to be similar to that estimated in the ice core samples from other such habitats (Abyzov et al., 1982; Sheridan et al., 2003). Present data represent only viable bacteria from the ice core and thus reveal only a part of the microbiome. Thus, there is a need to carry out more detailed studies to address the total biodiversity of the ice core using the metagenome approach. The ability of the microorganisms to survive prolonged periods of freezing and multiple cycles of freeze-thaw reflect the ability of psychrophiles and psychrotolerant bacteria to adapt to low temperature conditions. Bioprospecting studies indicated that these isolates could be used as a bioresource for the generation of psychrophilic enzymes. Earlier studies on culturable bacteria suggested that distribution of isolates at different depths in ice cores may correlate with specific past climatic as well as environmental conditions, such as the monsoon (Petit et al., 1999; Thompson et al., 2000; Zhang et al., 2006b), temperature, nutrient availability (Petit et al., 1999; Thompson et al., 2000; Xiang et al., 2005) and dust content (Zhang et al., 2002). Thus, to gain better insight into bacterial diversity in ice cores with respect to temporal and spatial variations, more comprehensive studies would need to be carried out using multiple ice cores, employing both culture- and molecularbased approaches (Zhang et al., 2008) and undertaking seasonal studies so as to understand the dynamics of deposition of organisms in ice.
Supplementary Table 1 Physiological characteristics of bacterial strains representative of the 25 bacterial groups isolated from the Antarctic ice core. Serial Strain ID number
Growth characteristics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
4e30 4e18 4e37 4e37 4e37 4e37 4e37 4e37 4e37 4e37 4e30 4e30 4e30 4e18 4e30 4e30 4e30 4e30 4e18 4e30 4e37 4e18 4e18 4e37 4e30
AIC1e3 AIC2e15 AIC2e22 AIC3e7 AIC3e9 AIC3e16 AIC3e19 AIC3e20 AIC4e13 AIC5e1 AIC5e18 AIC5e21 AIC5e24 AIC5e28 AIC6e5 AIC6e11 AIC6e14 AIC8e6 AIC9e14 AIC10e14 AIC10e21 AIC11e10 AIC11e11 AIC11e14 AIC11e28
Enzyme activities
pH range Amylase Esculinase Gelatinase Urease Casei-nase Cata-lase DNase Lipase Temperature NaCl tolerance range ( C) 4 C 18 C 4 C 18 C 4 C 18 C 4 C 18 C 18 C 18 C 18 C 18 C (M) 1 0.5 0 2 0 2 0 0.5 0.5 0.5 1 1 1 2 0.5 0.5 2 1 1 1.5 0.5 0.5 0.5 0.5 1
6e8 6e8 6e8 6e9 6e8 6e9 6e8 6e8 6e9 6e9 6e9 6e9 6e9 6e9 6e8 6e9 6e9 6e8 6e11 6e10 6e9 8e9 7e9 6e10 6e9
e e e e e e e þ e e e e e e e e e e e e e e e e e
e e þ e e e e e e þ e e þ e e e e e e e e e e e e
e þþþ þ e e þþþ e þþþ þþþ e þ e e e þþþ þþþ þþ e e e þþþ e e þþþ e
e þþ þþ þ e þþþ e þþþ þþþ þþþ þ þ e e þ þþ þ e þ e þþ e e þþ e
e e e e e e e e e e e e e e e e e e e e þ e e þ e
þ, Weakly positive; þþ, moderately positive; þþþ, highly positive; , negative; x, no growth.
þ þ e e þ e e e þ e e e e e e e e þ e e þ e e e e
e þ e þþþ e e þ e e e þþþ þþþ þþþ þþþ þþþ e e e þþþ e e þþþ þþþ þþþ e
þ e e þþþ e e e e þ e þþþ þþþ þþ þþþ þþþ þ e þ þþþ þþ e þþþ þþþ þþþ e
e e e e e e e þ e e e e e e e e e e e e e e e e e
þ þ w þ þ þ þ þ þ e þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ e e e e e e e e e e e e e e e e þ e e e e e e e
þþ þþ þþ e e e e þ þþ e þ e e e þþþ þþ e þ e þþþ þþ þ e e þþþ
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S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
Supplementary Table 2 Fatty acid profiles of the 11 representative Gram-negative bacterial strains among the 25 bacterial groups isolated from the 8 different depths (AIC-1 to 4; AIC-6; AIC-9e11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica. Fatty acid composition Saturated C12:0 C14:0 C16:0 C17:0 C18:0 C18:0 10-methyl C19:0 C20:0
AIC1e3
1.5 2.0 1.5
AIC2e15
26.8
11.6 3.9 9.2
AIC3e9
31.9
AIC4e13
AIC6e5
AIC6e11
AIC6e14
AIC9e14
11.1
23.7 12.7
4.8 4.4 6.1
2.4 2.0 1.3
4.9 13.7
6.4
6.1
1.7
3.5
44.5
24.9
9.2
11.1
15.6
Total Branched iso-C12:0 iso-C13:0 anteiso-C13:0 iso-C14:0 iso-C14:0 3OH iso-C15:0 anteiso-C15:1 anteiso-C15:0 iso-C16:0 anteiso-C16:0 iso-C17:0 iso-C 17:1 u10c anteiso-C17:0 anteiso-C17:1 u9c iso-C 18:0 2OH iso-C19:0 anteiso-C19:0 iso-C20:0 Summed feature 4a Summed feature 9a
5.0
Total Unsaturated * C13:1 C15:1 u5C C16:1 u9c C16:1 u11c C16:1 2OH C 17:1 u8c C18:1 u9c C18:1 2OH C18:03(u6c) (6,9,12) Summed feature 3a Summed feature 5a
92.5
36.0
24.7
47.5
AIC10e14
AIC11e14
0.5 6.3 21.0 0.7 0.5 23.75
10.1 3.6 13.2 5.9 4.8
5.5
11.1
5.7
35.2
2.1 2.2 52.8
41.9
24.2 4.4 1.5
5.9 1.0 2.4
1.5 2.8
20.0
3.7
48.3 2.3
22.6 3.3
5.5 1.4 4.6
4.4
16.4 0.1
2.2
4.2 1.9
6.6
4.8 18.6 4.1
1.4
4.8 3.2 5.8
6.5 1.9
4.9 8.8 66.3 8.2
1.4 2.4
0.2 3.0 2.9
3.2 2.0 2.2
0.4 5.3
2.3
3.0
0.2
3.9 3.4
1.6 0.7 0.3 0.9 1.0
59.3
1.1 3.9 5.9
0.3 2.0
8.7 53.7
0.3
24.8
42.7
26.5
69.1
92.0
44.0
6.4
8.2
2.8
2.6
0.2 0.6 0.4
3.2
3.2 2.4
0.1
2.5 22.8 5.9
6.8 0.9 1.6
2.5 13.1 62.2
10.5
1.2 3.4
11.4
6.9
6.0
1.5 12.4
26.1
24.0
AIC10e14 1.1 0.3
AIC11e14
9.2
Fatty acid composition Summed feature 6a Summed feature 7a Summed feature 8a
AIC1e3
Total
2.5
*
AIC 1e25
AIC 1e25
9.2
AIC2e15
AIC3e9
AIC4e13
11.8
3.6 13.5
12.7
74.1
27.6
42.6
AIC6e5
AIC6e11
AIC6e14
AIC9e14 3.9
17.9 29.1
6.0
2.8
19.3
42.6
44.1
Unsaturated includes C13:1, C15:1 u9c, C16:1 u9c, C16:1 u11c, C17:1 u8c, C18:1 u9c, C18:3 (6, 9, 12), summed feature 3, summed feature 6, summed feature 7 and summed feature 8. Iso- þ anteiso includes all the iso- and anteiso fatty acids indicated in the table. Saturated includes C12:0, C14:0, C16:0, C17:0, C18:0, C19:0 and C20:0. a Summed features represent groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 3-(C16:1 u7c/16:1 u6c); summed feature 4-(iso-C17:1 I/anteiso-C17:1 B); summed feature 5-(anteiso-C18:0/C18:2 u6,9c); summed feature 6-(C19:1 u11c/C19:1 u9c); summed feature 7-(C19:1 u7c/ C19:1 u6c); summed feature 8-(C18:1 u7c/C18:1 u6c); summed feature 9-(iso-C17:1 u9c/C16:0 10-methyl).
Supplementary Table 3 Fatty acid profiles of the 14 representative Gram-positive bacterial strains among the 25 bacterial groups isolated from the 5 different depths (AIC-2; AIC-3; AIC-5; AIC-10; AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica. Fatty acid composition Saturated C12:0 C12:0 3OH C14:0 C15:0 2OH C16:0 C17:0 2OH C17:0 10-methyl C18:0 C19:0 C20:0
Fatty acid composition iso-C17:0 iso-C17:0 3OH iso-C 17:1 u10c anteiso-C17:0 anteiso-C17:1 u9c iso-C18:1 H iso-C18:0 iso-C19:0 anteiso-C19:0 iso-C20:0 Summed Feature 2a Summed Feature 4a Summed Feature 9a Total
AIC 3e7
AIC 3e16
0.6 16.3
16.3
0.8
11.2
0.3
0.6
0.2
0.5
1.9
12.3
2 1
1.42 1.0 0.8
AIC 3e19 1.5 3.3 3.0 0.8 5.7 1.6
AIC 3e20
AIC 5e1
3.8 32.7
24.2
5.6
8.1 5.1 6.0
14.2 10.1 10.8
21.5
55.7
59.3
AIC 5e18
AIC 5e21
AIC 5e24
0.6
0.5
5.3
0.4
0.6
35.9
1.0
1.4
1.0 4.8
0.3 0.3
47.6
2.5
2.0
1.0
11.3 10.2 24.7
0.8 12.4
1.0 8.5
69.3 0.3 0.4
7.4
AIC 5e28
AIC 10e21
2.1
4.4
3.4
6.4
8.2
13.7
AIC3e7
AIC3e16
13.0
0.7
7.5
13.0
1.2 3.3
2.0 2.2
AIC 11e28
1.5
0.8
10.2
2.7
6.2
4.5
1.0
2.9
3.7 3.1 2.0
15.3
11.2
7.1
15.8
6.5
3.8
3.2
3.9
4.3
1.1
2.3 0.4
2.5
8.9
0.5
41.7
6.3
1.9
3.8
1.2 0.4
27.7 1.5 0.5
53.5 2.7
32.5 3.2
13.0
0.4 1.6
3.3
21.9
8.1
AIC3e19
4.8 2.0 2.5
AIC3e20
3.2 7.7
AIC5e1
6.3
8.5
AIC5e18
11.3 3.5
AIC5e21
AIC5e24
0.8
0.7
0.8 1.5 0.3
1.1 4.4
AIC10e21
AIC11e10
AIC11e11
19.2
3.0
9.9
1.8
15.9
6.2 13.6 35.7
1.5 5.0 4.3
49.0
AIC11e28
8.0 1.4 1.1
2.8
3.6 0.4 1.0
8.3
79.2
85.9
3.0 2.7
6.4 95.5
1.4
14.1
AIC5e28
1.2
3.7
83.6
1.0 41.1 9.5 1.6
1.9
11.4 0.4
AIC 11e11
0.9
1.0 AIC2e22
AIC 11e10
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
Total Branched iso-C12:0 anteiso-C12:0 iso-C13:0 anteiso-C13:0 iso-C14:0 iso-C14:0 3OH anteiso-C14:0 iso-C15:0 iso-C15:1 anteiso-C15:1 anteiso-C15:0 iso-C16:0 iso-C16:1 u11c anteiso-C16:0
AIC 2e22
13.9
36.1
22.9
28.5
79.6
79.5
81.0
27.8
8.8 79
(continued on next page)
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Supplementary Table 3 (continued ) Fatty acid composition
AIC 2e22
Unsaturated * C12:1 3OH C15:1 u6c C15:1 u5c C16:1 u9c C16:1 u7c alcohol C16:1 u11c C 17:1 u6c C18:1 C18:1 u9c C18:1 u7c 11-methyl C18:3 u6,9,12c C20:2 u6,9C C20:1 u9c C20:1 u7c Summed Feature 3a Summed Feature 5a
AIC 3e16
AIC 3e19
AIC 3e20
AIC 5e1
AIC 5e18
AIC 5e21
AIC 5e24
AIC 5e28
AIC 10e21
AIC 11e10
AIC 11e11
6.1
2.3 53.9
AIC 11e28
1.1 2.5 1.5
1.9
6.2 0.3
1.2
1.1 0.4
7.0 3.5
1.4
4.0 14.7 0.6
9.8
5.5
0.5
1.5
8.1 1.0 1.0
0.3 0.2
8.0
2.0
0.4 AIC 2e22
Total
0
11.9
12.7 1.9
AIC 3e7
AIC 3e16
AIC 3e19
0.2
11.2
26.6
2.2
31.3
56.9
0.4 11.2 0.3
0.2
4.2 1.1
1.1 2.2
AIC 3e20 3.6
AIC 5e1
AIC 5e18 3.2 0.9
AIC 5e21 0.8 0.2
AIC 5e24
AIC 5e28 4.0
AIC 10e21
AIC 11e10
AIC 11e11
AIC 11e28 39.2 23.7
3.6
17.8
24.2
17.9
11.6
5.9
2.2
7.7
58.1
70.1
Unsaturated includes C13:1, C15:1 u9c, C16:1 u9c, C16:1 u11c, C17:1 u8c, C18:1 u9c, C18:3 (6, 9, 12), summed feature 3, summed feature 6, summed feature 7 and summed feature 8. Iso- þ anteiso includes all the iso- and anteiso fatty acids indicated in the table. Saturated includes C12:0, C14:0, C16:0, C17:0, C18:0, C19:0 and C20:0. a Summed features represent groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 2-(iso-C16:1 I/C14:0 3OH); summed feature 3-(C16:1 u7c/16:1 u6c); summed feature 4-(iso-C17:1 I/anteiso-C17:1 B); summed feature 5-(anteiso-C18:0/C18:2 u6,9c); summed feature 7-(C19:1 u7c/C19:1 u6c); summed feature 8-(C18:1 u7c/C18:1 u6c); summed feature 9-(iso-C17:1 u9c/C16:0 10methyl).
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
0.7
Fatty acid composition Summed feature 7a Summed feature 8a
*
AIC 3e7
S. Shivaji et al. / Research in Microbiology 164 (2013) 70e82
81
Supplementary Table 4 Affiliation of the 138 bacterial strains, isolated from the 11 different depths (AIC-1 to AIC-11) of the Antarctic ice core collected from the continental ice sheet at central Dronning Maud Land, Antarctica, to phyla, genera and species/subspecies based on analysis of complete 16S rRNA gene sequences. Phylum
Genus
Species/subspecies
AIC samples 1
Actinobacteria
Firmicutes
Arthrobacter Brevibacterium Rhodococcus
Bacillus Brevibacillus Paenibacillus
Sporosarcina Staphylococcus Bacteroidetes Proteobacteria
Flavobacterium Sphingobacterium Janthinobacterium Naxibacter Pseudomonas
Sphingomonas Stenotrophomonas
Arthrobacter rhombi Brevibacterium antiquum Rhodococcus cercidiphylli Rhodococcus fascians Rhodococcus qingshengii Bacillus psychrodurans Bacillus stratosphericus Brevibacillus centrosporus Brevibacillus panacihumi Paenibacillus humicus Paenibacillus peoriae Paenibacillus xylanexedens Sporosarcina globispora Staphylococcus sciuri subsp. sciuri Flavobacterium pectinovorum Sphingobacterium anhuiense Janthinobacterium lividum Naxibacter haematophilus Pseudomonas azotoformans Pseudomonas brenneri Pseudomonas poae Pseudomonas stutzeri Sphingomonas aerolata Sphingomonas faeni Stenotrophomonas rhizophila
2
1 2
Total
3
4
5 1 1 2 2 1
1
6
7
8
9
10
7 1
11
3
1 1 1
1 1 6 4
3
3
3 1 1 1 1 2
2 6
4
1
3 1
1
24
11
3 5
4
1 1 4
10
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