Vertical distribution of bacteria in a lake sediment from Antarctica by culture-independent and culture-dependent approaches

Research in Microbiology 162 (2011) 191e203 www.elsevier.com/locate/resmic

Vertical distribution of bacteria in a lake sediment from Antarctica by culture-independent and culture-dependent approaches Sisinthy Shivaji a,*, Kiran Kumari a, Kankipati Hara Kishore a, Pavan Kumar Pindi a, Pasupuleti Sreenivasa Rao a, Tanuku Naga Radha Srinivas a, Rajesh Asthana b, Rasik Ravindra c a

Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India b Antarctic Division, Geological Survey of India, Faridabad 121 001, India c National Center for Antarctic & Ocean Research, Headland Sada, Vasco-da-Gama, Goa 403 804, India Received 12 March 2010; accepted 21 September 2010 Available online 30 November 2010

Abstract Bacterial diversity of the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of a 136-cm-long sediment core sampled from a freshwater lake in Antarctica was determined by the culturable approach, T-RFLP and 16S rRNA gene clone libraries. Using the culturable approach, 41 strains were isolated and, based on phylogenetic analysis, they could be categorized into 14 groups. Representatives of the 14 groups varied in their growth temperature range (4e30
C), in their tolerance to NaCl (0e2 M NaCl) and in the growth pH range (5e11). Eleven of fourteen representative strains exhibited either amylase, lipase, protease and (or) urease activities at 4
C. Bacterial diversity at the phyla level using T-RFLP and 16S rRNA clone libraries was similar and clones were affiliated with Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. TRFs affiliated with Spirochaetes were detected only by the T-RFLP approach and clones affiliated with Caldiserica only in the clone libraries. Stratification of bacteria along the depth of the sediment was observed both with the T-RFLP and the 16S rRNA gene clone library methods, and results indicated that stratification was dependent on the nature of the organism, aerobic or anaerobic. For instance, aerobic Janthinobacterium and Polaromonas were confined to the surface of the sediment, whereas anaerobic Caldisericum was present only in the bottom portion of the core. It may be concluded that the bacterial diversity of an Antarctic lake sediment core sample varies throughout the length of the core depending on the oxiceanoxic conditions of the sediment. Furthermore, these psychrophilic bacteria, due to their ability to produce extracellular cold active enzymes, might play a key role in the transformation of complex organic compounds. Ó 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: T-RFLP; 16S rRNA clone library; Culturable approach

1. Introduction In freshwater sediments, prokaryotes play a major role in the transformation of complex organic compounds and minerals (Jurgens et al., 2000). Consequently, investigating microbial structure and function in freshwater sediments is of immense importance so as to understand the role of microorganisms in * Corresponding author. Tel.: þ91 40 27192504; fax: þ91 40 27160311. E-mail addresses: [email protected] (S. Shivaji), [email protected] (K. Kumari), [email protected] (K.H. Kishore), pavankumarpindi@ gmail.com (P.K. Pindi), [email protected] (P.S. Rao), [email protected] (T.N. Radha Srinivas), [email protected] (R. Ravindra).

aquatic ecosystems. Antarctic freshwater lakes are relatively simple aquatic ecosystems, with low species diversity, low species richness, restricted environmental variables and short food chains. Other characteristics of Antarctic freshwater lakes are constant low temperatures, very low biomass and limited trophic complexity (Ellis-Evans, 1996). Approaches to investigating microbial ecosystems in freshwater sediments on the basis of conventional culture methods are important since, via this approach, the ecological role of the cultivated and characterized prokaryotes can be estimated. However, using this approach, only a miniscule fraction of the bacteria get cultivated, leaving a vast majority uncultivated. Culture-independent molecular approaches based on small subunit rRNA have also been used for

0923-2508/$ - see front matter Ó 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2010.09.020

192

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

studies of microbial ecology in freshwater sediments (Altmann et al., 2003; Purdy et al., 2003). However, information on microbial composition in freshwater sediments is limited, since previous studies have mainly focused on certain functional groups such as nitrifiers, denitrifiers, sulfate reducers, methanogens and methanotrophs (Costello et al., 2002; Gregory et al., 2003; Purdy et al., 2003). Terminal restriction length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene has become a technique of choice in environmental microbiology, since it provides information on microbial communities with respect to their dynamics, diversity and richness in a range of environments (Moeseneder et al., 1999; Luna et al., 2004). Further, T-RFLP provides a facile means of assessing changes in microbial community structure on temporal and spatial scales (Mummey and Stahl, 2003). T-RFLP coupled with 16S rRNA gene clone library construction and sequencing would help to validate the information on the composition of microbial communities (Schu¨tte et al., 2008). Variations in bacterial population structure along the depth of a sediment have been previously reported in the coastal Pacific (Urakawa et al., 2000), for cold seep sediments (Inagaki et al., 2002) and also in Antarctic sediments (Bowman and McCuaig, 2003; Bowman et al., 2003; Li et al., 2006; Sjo¨ling and Cowan, 2003). Those studies indicated that maximum bacterial diversity was predominant in the subsurface sediment layers. Most of those studies in the Antarctic were confined to a specific region and used just one approach (TRFLP, DGGE, 16S rRNA gene library or culturable bacteria approach) (Li et al., 2006; Mummey and Stahl, 2003; Urakawa et al., 2000) or, at times, a combination of two approaches (Moeseneder et al., 1999; Schu¨tte et al., 2008). A combination of two or more approaches is likely to provide a more comprehensive picture of bacterial diversity in an environmental sample, since there is a likelihood that limitation of one approach could be overcome by another approach. For instance, the inability to culture all bacteria in a sample could be overcome by the 16S rRNA approach. In the present study, the bacterial diversity of sediment from a freshwater lake in Schirmacher Oasis, Queen Maud Land, Antarctica was studied using a combination of TRFLP with a 16S rRNA clone library and the culturable approach. The objectives were to assess total bacterial diversity of lake sediment, to monitor stratification of the bacteria along the depth of a sediment core, to evaluate whether stratification was dependent on oxiceanoxic conditions and to evaluate their extracellular enzyme activities with respect to degradation of proteins, lipids, polysaccharides and urea.

was then sampled by manually hammering a steel pipe (5 cm inner diameter) into which a PVC pipe was inserted into the sediment. The core that was collected was gently pushed out from the PVC pipe and the 136 cm long core was cut into sections of defined length. The upper part of the core up to 16 cm was covered with algal mats; it was very loose, not compact and could not be cut into sections. However, the remaining length of the core from 18 to 136 cm was compact and could be conveniently cut into 4 cm sections. Three samples, subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm), were analyzed during this study. The lake water and sediment temperatures at the time of sample collection were 2 to 1.5 and 4e5
C respectively. Lake 6 is a melt water lake which receives inflows from the surrounding hills. The depth of the lake varies from 2.5 to 6.0 m in the deepest part. The core was from the deepest part of the lake. 2.2. Isolation, culturing and abundance of bacteria Approximately 100 mg of the sediment sample was suspended in 900 ml of sterile water and subjected to shaking for 2 h at 10
C. The supernatant was serially diluted and 100 ml was plated 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 both 4 and 10
C for 15 days (Shivaji et al., 1992). The number of colony-forming units was recorded and different morphotypes from each depth were purified and maintained on ABM plates. Total bacterial count in the sediment samples was done in triplicate by epifluorescence microscopy using the BacLight_Bacterial Viability kit (Invitrogen, Oregon, USA). Also for this purpose, 100 mg of the sediment sample was suspended in 900 ml of sterile water and subjected to shaking for 2 h at 10
C. The tube was then allowed to stand at 10
C for the sediment to settle. To 100 ml of the above sample, 0.3 ml of the SYTO 9 and propidium iodide stains were added, mixed gently and incubated on ice till observed under the microscope. The tube was wrapped with aluminum foil to avoid direct light. An aliquot of the bacterial suspension was then transferred to the Petroff-Hausser counting chamber and bacteria were counted using a fluorescent microscope (Axioplan 2, Zeiss, Germany). The number of bacteria per ml was then calculated by multiplying the number counted by the dilution factor and by extrapolating the volume to 1 ml and then to 1 g soil. 2.3. Characterization of the bacterial strains

2. Materials and methods 2.1. Sampling site and sample collection A sediment sample was collected from a freshwater lake, designated lake 6 (L-6), in the winter season of 2005, when 3e4 feet of the surface was frozen, The lake is located in Schirmacher Oasis (70
46’ 04’’-70
44’ 21’’ S; 11
49’ 54’’-11
26’ 030 ’ E), Queen Maud Land, Antarctica. The average annual temperature of the oasis is 10
C and mean wind velocity is 10 m/s. The surface ice was broken with an axe and the sediment of L-6

The growth of the bacterial strains at different temperatures, pH and salt was checked using ABM agar plates (Shivaji et al., 1992). The extracellular enzymatic activities such as amylase, lipase, protease and urease were checked by streaking the culture on ABM plates supplemented with 0.2% soluble starch for amylase activity, 1% Tween-60 along with 0.01% CaCl2 for lipase activity, 0.3% casein for protease activity and 10% filtered urea for urease activity, respectively and incubating at 4
C (Srinivas et al., 2009). DNA was isolated from all cultures, and the 16S rRNA gene was amplified

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

and sequenced as described earlier (Shivaji et al., 2009; Srinivas et al., 2009). 2.4. T-RFLP analysis of 16S rRNA genes Total genomic DNA was extracted from 2.5 g of sediment sample from a particular depth (18e22 cm, 60e64 cm and 100e104 cm respectively), according to the method described by Yeates et al. (1998). DNA concentrations were determined by measuring the absorbance at 260 nm and purity was checked by determining 260/280 nm absorbance ratio. The bacterial 16S rRNA gene was amplified from the genomic DNA by PCR using the primer set 16S1 50 -FAM-GAG TTT GAT CCT GGC TCA-30 and 16S2 50 -ACG GCT ACC TTG TTA CGA CTT-30 under the PCR conditions described earlier (Shivaji et al., 2009; Pradhan et al., 2010). For all three samples, two independent PCR reactions (50 ml) were performed. Subsequently, the amplified products were pooled (Schu¨tte et al., 2008), purified with a Quiaquick PCR purification kit (Qiagen Inc.,Chatsworth, USA) and digested in a 20 ml reaction volume with 10 U of RsaI at 37
C for 3 h (Luna et al., 2004). Three microliters of each of the digested samples were mixed with 6.7 ml of de-ionized formamide, 0.3 ml of internal size standard (GS400-ROX; Applied Biosystems, United Kingdom) and then denatured at 94
C for 2 min and kept on ice for 5 min. Fragments were analyzed in GeneScan mode in an ABI Prism 3730 Genetic Analyzer (Applied Biosystems, United Kingdom). T-RFLP analysis was carried out 3 times for each sample and the results were consistent. 2.5. Data analysis T-RFLP profiles were produced using the GeneMapper software (version 3.7; ABI, United Kingdom), and peaks in the range of 50e500 bp and with intensities 50 fluorescence units were manually aligned. The relative abundance of each TRF was calculated as a percentage of the total height summed over all peaks (Covert and Moran, 2001). The fragments that differed by less than 2 base pairs in different profiles were considered identical (Li et al., 2007). Phylogenetic assignment was performed using a web-based analysis tool (PATþ) provided by MiCA3 (http://mica.ibest. uidaho.edu) based on the RDP Release 9.60 16S rRNA gene database (Shyu et al., 2007; Nakano et al., 2008) and phylogenetic assignment tool (PAT) (http://trflp.limnology.wisc.edu/ assignment) (Kent et al., 2003). The phylogenetic affiliation of the TRFs was further validated by in silico restriction digestion using TriFLe (Junier et al., 2008) (http://cegg.unige.ch/ trifle/trifle.jnlp) and NEB cutter.

193

the pMOS Blue Blunt End vector system (Amersham Biosciences, New Jersey, USA) following the instructions in the manual. Transformants were selected on an LB agar plate containing 20 mg/ml X-gal and 60 mg/ml ampicillin and incubated at 37
C overnight. 2.7. PCR amplification of the 16S rRNA gene, sequencing and phylogenetic analysis The 16S rRNA gene was amplified from the transformants by colony PCR using the vector-targeted M13 forward (50 GTA AAA CGA CGG CCA GT-30 ) and M13 reverse (50 -GGA AAC AGC TAT GAC CAT G-30 ) primers, respectively, and sequenced using the primers M13 forward, M13 reverse, pD (50 -CAG CAG CCG CGG TAA TAC-30 ) and pF* (50 -ACG AGC TGA CGA CAG CCA TG-30 ) (Pradhan et al., 2010; Reddy et al., 2010). Vector-based sequences and chimeric sequences were eliminated using Gene Tool version 2 (www. biotools.com). Sequences were then subjected to BLAST to identify the nearest taxa and aligned with sequences belonging to the nearest taxa, (http://.www.ncbi.nlm.nih.gov) using Clustal X, and phylogenetic trees were constructed using the Neighbor joining method (Pradhan et al., 2010). Bootstrap analysis, based on 1 000 replicate datasets, was performed to assess stability among the clades. 2.8. Statistical analyses of the cloned libraries 16S rRNA gene sequences of clones showing 97% sequence similarity were grouped into the same OTU (phylotype) (Pradhan et al., 2010). The ShannoneWiener Diversity Index (http://www.changbioscience.com/genetics/shannon. html) was used to calculate the Shannon index (H/), Evenness (J/) and Simpson’s index (D). Rarefaction analysis was done using the site Online Calculation (http://biome.sdsu.edu/ fastgroup/cal_tools.htm). Coverage of 16S rRNA gene clone libraries was calculated as described previously (Pradhan et al., 2010). Rarefaction curves were generated to compare the relative diversity and coverage of each library. Principal component analysis (PCA) was performed using the SPSS statistical computing package (version 16.0; SPSS, Inc., Chicago, IL, USA) and was employed to group or to separate samples based on biogeochemical parameters [depth, total bacterial count, organic carbon, Li, Be, Na, Mg, Al, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Rb, Sr, Ag, Cd, Te, Ba, Tl, Pb and Bi] in each sample. 2.9. Nucleotide sequence accession numbers

2.6. Construction of the 16S rRNA gene library The 16S rRNA gene was amplified using the two primers 16S3 (50 -TCC TAC GGG AGG CAG CAG-30 ) and 16S4 (50 GGC GGT GTG TAC AAG GCC C-30 ) as described earlier (Pradhan et al., 2010) and the amplified DNA fragments purified as above using Qiagen gel columns, and cloned into

All sequences of the 16S rRNA gene clone library were deposited in GenBank with accession numbers GU000065GU000299, GU000425, GU000426, GU000428, GU000429 and GU000439-GU000496. All 16S rRNA gene sequences of the isolated strains were deposited in GenBank with accession numbers GU244354-GU244367 and GU733455-GU733481.

194

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

3. Results 3.1. Bacterial abundance and viable bacteria of the Antarctica lake sediment Soil texture varied from top to bottom and was observed to be sandy in the subsurface sediment, whereas it was made up of fine silt at the bottom (Table 1). The total bacterial count in the sediment sample from lake 6 (L-6), Schirmacher Oasis, Antarctica, from the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of the sediment core was 5.7e7.9  107 bacteria g1 of sediment (Table 1). In contrast, as anticipated, the viable bacterial count was less and was observed to be between 11.0, 5.2 and 9.6  103 cfu g1 sediment, from the top to the bottom of the sediment. The organic carbon level and the presence of other elements like Li, Be, Na, Mg, Al, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Rb, Sr, Ag, Cd, Te, Ba, Tl, Pb and Bi from the top to the bottom of the sediment varied slightly (Table 1). The 41 strains isolated from the subsurface, middle and bottom of the sediment of Lake 6, based on 16S rRNA gene sequence of the strains, indicated that they belonged to the genera Janthinobacterium, Pseudomonas, Flavobacterium, Arthrobacter, Brevibacterium, Cryobacterium, Microbacterium and Paenisporosarcina (Table 2). Isolates exhibiting 97% or

<97% similarity with the nearest phylogenetic neighbor at the 16S rRNA gene sequence level were considered as one group and those which exhibited >97% similarity were grouped into one group. Using this criterion, the 41 strains could be grouped into 14 groups (Table 2) and were used for all subsequent studies. Phylogenetic analysis based on the 16S rRNA gene sequence of the 14 representative isolates of the 41 culturable isolates (Supplementary Fig. 1) confirmed their affiliation with the nearest phylogenetic neighbor, as indicated by BLAST of the 16S rRNA gene sequence (Table 2). 3.2. Characterization of the bacterial strains The representative isolates from each of the 14 groups were either psychrophilic (growth between 4 and 20
C) or psychrotolerant (growth between 4 and 30
C), did not require NaCl in the medium and the majority were able to tolerate 0.5e2.0 M NaCl (Table 3). A few of the strains could also grow at a wide pH range of 5e11 and a few in the narrow range of 6e9 (Table 3). Furthermore, out of 14 strains, 6, 4, 6 and 3 strains showed amylase, lipase, protease and urease activities, respectively, at 4
C (Table 3). None of the strains exhibited all the enzyme activities studied, though Lc30-1 showed 3 out of the 4 enzyme activities (Table 3). 3.3. T-RFLP profiles

Table 1 Physico-chemical characteristics and total bacterial count of the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of sediment of Lake 6, Schirmacher Oasis, Antarctica. Soil parameters

Subsurface

Middle

Bottom

Soil texture Depth (cm) Total bacterial count/g sediment (107) Organic carbon (%) Li (ppm) Be (ppm) Na (ppm) Mg (ppm) Al (ppm) K (ppm) Ca (ppm) V (ppm) Cr (ppm) Mn (ppm) Fe (ppm) Co (ppm) Ni (ppm) Cu (ppm) Zn (ppm) Ga (ppm) Rb (ppm) Sr (ppm) Ag (ppm) Cd (ppm) Te (ppm) Ba (ppm) Tl (ppm) Pb (ppm) Bi (ppm)

Sandy silt 18e22 7.9

Silt dominant 60e64 7.4

Fine silt 100e104 5.7

16.2 28.1 1.0 1489.6 4869.2 12140.0 3164.4 2757.5 134.5 86.8 485.9 62076.3 26.0 59.3 2262.1 249.0 25.5 112.3 127.7 0.3 0.1 0.1 613.4 0.7 13.7 0.1

16.2 28.5 1.1 1778.0 5108.4 13147.5 3297.8 3116.7 142.6 83.1 526.3 62818.0 26.9 55.4 179.9 259.2 25.5 110.2 139.1 0.2 0.1 0.1 587.8 0.7 12.1 0.1

8.9 37.4 1.4 2110.5 5177.6 14221.7 3920.3 3798.4 173.7 96.1 610.4 79109.7 30.8 62.5 71.5 318.6 33.4 148.6 187.1 0.2 0.1 0.1 787.3 0.9 16.5 0.1

The soil samples from the subsurface, middle and bottom of the sediment yielded about 20e40 mg DNA g1 of soil. T-RFLP analysis of the sediment from L-6 was attempted only for the subsurface, middle and bottom of the sediment sample, since the surface of the sediment up to 16 cm appeared to be loose and full of vegetation and could not be cut into sections. T-RFLP profiles for the three depths were very similar (Fig. 1). The total number of terminal restriction fragments (TRFs) observed at all 3 depths was 38, and they represented 19 different TRFs (Table 3). Seven TRFs (119, 142, 290, 309e310, 427, 440e442 and 457 bp respectively) were common to all depths, 5 TRFs were shared between two depths (109, 147e148, 419, 430 and 437 bp respectively) and the remaining 7 were unique TRFs (208, 301, 394, 400, 453, 467 and 474 bp respectively) which were present only at one particular depth. The distribution and quantity of each TRF in the sediment is shown in Table 3. T-RFLP analysis was carried out 3 times for each sample and the results were consistent. 3.4. Phylogenetic assignment of TRFs TRFs generated were assigned to phyla, probable genus and species in some cases (Table 4). The presumptive bacterial identification was also validated by in silico restriction digestion of 16S rRNA of the identified bacterium which yielded TRFs identical to the results in Table 4. For instance, the TRF of 290 bp was identified by a web-based tool as Paenibacillus illinoisensis, and following in silico analysis of the 16S rRNA gene of P. illinoisensis, a TRF of 290 bp was obtained, thus confirming the presumptive identification (Table 4).

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

195

Table 2 Tentative identification of the 41 bacterial strains isolated from the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of the sediment of Lake 6, Schirmacher Oasis, Antarctica, based on BLAST analysis of the 16S rRNA gene sequences. Serial number

Strain number

Nearest phylogenetic neighbor and 16S rRNA gene sequence similarity (%)

Lc10-8; Lc10-10; Lc30-2; Lc50-4; Lc51-1; Lc51-4; Lc51-5; Lc51-6

Janthinobacterium lividum DSM 1522T Y08846 (99.7)

Lc10-7; Lc10-9; Lc31-3; Lc31-6 Lc10-2 Lc31-2; Lc31-4 Lc1-1; Lc1-2

Pseudomonas Pseudomonas Pseudomonas Pseudomonas

Lc50-3

Flavobacterium psychrolimnae LMG22018T AJ585428 (99.5)

Lc5-1; Lc10-5; Lc30-3 Lc10-1; Lc31-7; Lc51-7 Lc30-1; Lc51-2 Lc10-4; Lc10-6 Lc31-1; Lc31-5; Lc31-9; Lc31-10; Lc31-11; Lc31-12; Lc51-8 Lc30-4; Lc50-1 Lc1-3

Arthrobacter oxydans DSM 20119T  83408 (99.1) Arthrobacter scleromae YH-2001T AF330692 (99.2) Arthrobacter sulfonivorans ALLT AF235091 (99.2) Brevibacterium antiquum VKM Ac-2118T AY243344 (98.4) Cryobacterium psychrotolerans 0549T DQ515963 (97.6)

Lc5-3; Lc10-3; Lc50-2

Paenisporosarcina macmurdoensis CMS 21wT AJ514408 (99.7)

Proteobacteria Betaproteobacteria 1 Gammaproteobacteria 2 3 4 5 Bacteroidetes Flavobacteria 6 Actinobacteria 7 8 9 10 11 12 13 Firmicutes Bacilli 14

azotoformans IAM1603T D84009 (99.7) frederiksbergensis JAJ28T AJ249382 (99.9) gessardii CIP 105469T AF074384 (99.7) mandelii CIP 105273T AF058286 (100)

Cryobacterium roopkundense RuGl7T EF467640 (98.9) Microbacterium profundi Shh49T EF623999 (99.9)

The accession numbers of the 14 representative strains (indicated in bold) are GU244354-GU244358, GU244360-GU244362, GU244364, GU244365, GU733456, GU733457, GU733469 and GU733480.

3.5. 16S rRNA gene clone libraries About 200 ng of genomic DNA from the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of the sediment sample of Lake 6 was used for construction of three 16S rRNA gene libraries which were designated as 18 cm, 60 cm and 100 cm libraries respectively. The number of clones in the 18, 60 and 100 cm libraries was 132, 101 and 64, respectively, with an insert size of approximately 1 kb. Based on BLAST analysis, clones from the 18 cm library could be categorized into five major phylogenetic groups,

namely the Gammaproteobacteria (62.1%), Betaproteobacteria (6.8%), Bacteroidetes (28.8%), Firmicutes (0.8%) and Actinobacteria (1.5%) (Table 5 and Supplementary Table 1). But with an increase in depth in the 60 cm library, only Gammaproteobacteria (99%) and Bacteroidetes (1.0%) persisted. In contrast, in the 100 cm library, both Gammaproteobacteria and Betaproteobacteria were absent and the most dominant were clones affiliated with Caldiserica (82.8%) (Table 5 and Supplementary Table 1). At the genus level, 16S rRNA gene clones affiliated with the genera Pseudomonas (88e99% similarity) and Flavobacterium

Table 3 Phenotypic characteristics of the representative isolates from each of the 14 groups of bacterial strains isolated from the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of the sediment of Lake 6, Schirmacher Oasis, Antarctica. Serial number

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Sample ID Lc1-2 Lc1-3 Lc10-2 Lc10-3 Lc10-5 Lc10-6 Lc10-7 Lc30-1 Lc30-2 Lc31-4 Lc31-5 Lc50-1 Lc50-3 Lc51-7

Enzymatic activity (4
C)

Growth characteristics Temp. range (
C) 4e30 4e20 4e20 4e30 4e20 4e30 4e30 4e30 4e20 4e30 4e20 4e20 4e30 4e20

NaCl tolerance (M) 0.5 1.0 0.5 0.5 0.5 2.0 1.0 0.5 0.5 0 0.5 0.5 0.5 0

pH range 5e10 6e11 6e9 6e11 6e11 5e11 5e11 5e11 6e9 5e9 6e11 6e11 6e11 6e9

Amylase e e þ þ e e þ þ e þ e e þ e

Lipase þ e e e þ e þ þ e e e e e e

Protease e e e þ e e e þ þ e þ þ þ e

Urease e e þ e þ e e e e e þ e e e

196

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

Fig. 1. Electropherograms of the 50 terminal restriction fragments of RsaI-digested 16S rRNA amplified from the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of a sediment from Lake 6 in Schirmacher Oasis, Antarctica.

(96e99% similarity) constituted 61 and 28% of the total number of clones, respectively, in the 18 cm library. The remaining clones were affiliated with the genus Janthinobacterium (5%) and 6 other genera (Supplementary Table 1). In the 60 cm library, 99% of the clones were affiliated with Pseudomonas (90e99% similarity) and the remaining clone in the library was similar to the genus Flavobacterium (1%). The clones in the 100 cm library appeared to be totally different from the 60 cm library and resembled the 18 cm library only with respect to two clones belonging to the genera Streptomyces and Paenisporosarcina (Supplementary Table 1). The 100 cm library was dominated by clones affiliated with the phylum Caldiserica and a few clones were related to 5 genera in Firmicutes which constituted 9.3% of the library, while the remaining clones were affiliated with the genera Streptomyces, Sporichthya, Paenisporosarcina and Thermobaculum (Supplementary Table 1). 3.6. Phylogenetic analysis of 16S rRNA gene clones Phylogenetic analysis indicated that most of the clones clustered with their nearest phylogenetic neighbor (Figs. 2 and 3). However, a few of the clones did differ from their phylogenetic affiliation (Supplementary Table 1, Figs. 2 and 3). For instance, clone sequences affiliated with the two classes of phylum Proteobacteria (Fig. 2) clustered with their related sequences (Fig. 2), except for 3 clone sequences (L6B-52, L6B-127 and L6B-151). Clones L6B-52 and L6B-151 did not cluster with Pseudomonas veronii DSM 11331T or Pseudomonas umsongensis Ps 3-10T, the nearest phylogenetic neighbors, respectively but branched from Bacteroidetes (Fig. 2). Clone L6B-127 showed 93% pairwise sequence similarity with Janthinobacterium lividum DSM 1522T

(Supplementary Table 1), but formed an out group to both the Polaromonas and Janthinobacterium clusters (Fig. 2). All the clones related to Bacteroidetes and Actinobacteria formed clusters (Fig. 2) along with the reported strains of the genera (Supplementary Table 1). Clones affiliated with Caldiserica (53 clones) formed a robust clade with the Caldisericum exile AZM16c01T (Fig. 3). In Firmicutes, except for two clones (L6B-381 and L6B-389) which formed a separate branch and clustered with the Caldiserica cluster (Fig. 3), all the remaining clones clustered with their related sequences (Fig. 3). Clone L6B-364, which was identified as an unclassified bacterium based on BLAST analysis (Supplementary Table 1), clustered with Thermobaculum terrenum ATCC BAA-798T (Fig. 3). 3.7. Statistical analysis of 16S rRNA gene libraries In the three 16S rRNA gene libraries from the subsurface, middle and bottom of the sediment sample of lake 6, species richness was 17, 5 and 12, respectively, and the diversity coverage was 87.1, 95.0 and 82.8%, respectively. The rarefaction curves for the 3 libraries indicated that the clones were representative of each of the samples, as evidenced by the tendency of the curves to plateau (Supplementary Fig. 2). These observations are also supported by bacterial diversity parameters such as evenness (J’) (0.5, 0.14 and 0.4), Shannon index (H’) (1.5. 0.2 and 0.9) and Simpson’s index (D) (0.39, 0.92 and 0.66), respectively, for subsurface, middle and bottom of the sediment of lake 6. PCA based on biogeochemical properties, depth and total bacterial count of the subsurface, middle and bottom of the sediment sample of lake 6 are shown in Supplementary Fig. 3. The principal component factors 1 and 2 (PC1-99.955% and

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

197

Table 4 Distribution, quantitation and presumptive identification of TRFs following RsaI digestion of 16S rRNA genes from the subsurface, middle and bottom of the sediment of Lake 6, Schirmacher Oasis, Antarctica, up to the genus/species level based on species match by MiCA and in silico confirmation based on 16S rRNA gene from the database using Trifle/NEB. TRF (bp)

Relative abundance (%) Subsurface of sediment core (18e22 cm)

Middle of sediment core (60e64 cm)

Bottom of sediment core (100e104 cm)

109 119 142 147e148 208 290 301 309e310

39.0 5.4 0.8 e e 36.0 2.1 0.2

2.6 22.2 4.7 1.8 5.4 1.3 e 6.9

e 68.5 1.4 0.3 e 0.7 e 3.1

394* 400* 419 427

e 0.8 e 1.5

e e 3.5 30.3

0.7 e 0.6 3.6

430 437* 440e442

5.6 e 6.5

e 1.9 2.2

6.5 3.3 0.3

453

e

e

0.3 8.3

457

0.5

16.8

467

0.9

e

474

e

e

e

2.2

Species match by MiCA: and confirmed by in silico analysis using Trifle/NEB

Phylum

Rhodopseudomonas palustris strain GH Janthinobacterium lividum strain GA01 Alkaliphilus metalliredigens strain QYMF Uncultured rumen bacterium strain F24-A02 Uncultured rumen bacterium strain 4C0d-12 Paenibacillus illinoisensis strain 14 NRRL NRS-1356T Uncultured bacterium clone SJA-69 Flavobacteriaceae bacterium strain CNU041, Pedobacter sp. strain DL3 Not identified Not identified Polaromonas naphthalenivorans strain CJ2 Comamonas testosteroni RH 1104 ATCC 11996T, Craurococcus roseus strain NS130, Enterobacter pyrinus KCTC 2520T Alcaligenes faecalis subsp. faecalis strain M3A Not identified Pseudomonas sp. lip23, Acinetobacter septicus AK001 0106 Streptomyces sp. sd-45, Cellulosimicrobium sp. TUT1222, Cryobacterium sp. DR9 Streptomyces armeniacus JCM 3070T, Eubacterium lentum JCM 9979, Brevibacterium celere KMM3637, Arthrobacter globiformis 168 DSM 20124T Thermoanaerobacter sp. X514, Tissierella praeacuta ATCC 25539T Thermomonospora chromogena

Proteobacteria Proteobacteria Firmicutes Firmicutes Firmicutes Firmicutes Spirochaetes Bacteroidetes e e Proteobacteria Proteobacteria

Proteobacteria e Proteobacteria Actinobacteria Actinobacteria

Firmicutes Actinobacteria

*Out of 19 TRFs, only three marked with an asterisk could not be identified either by MiCA or by in silico analysis.

Table 5 Comparison of 16S rRNA gene clone libraries constructed using the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of a sediment sample of Lake 6, Schirmacher Oasis, Antarctica. Phylum/Class

16S rRNA gene Library 18e22 cm sediment

Proteobacteria Betaproteobacteria Gammaproteobacteria Bacteroidetes Flavobacteria Actinobacteria Actinobacteria Caldiserica Caldiserica Firmicutes Bacilli Clostridia Unclassified

60e64 cm sediment

100e104 cm sediment

Number of clones

Library (%)

Number of clones

Library (%)

Number of clones

Library (%)

9 82

6.8 62.1

0 100

0 99.0

0 0

0 0

38

28.8

01

1.0

0

0

2

1.5

0

0

3

4.7

0

0

0

0

53

82.8

1 0 0

0.8 0 0

0 0 0

0 0 0

1 6 1

1.6 9.3 1.6

198

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

91

61

23

L6B-133 Thioalcalovibrio denitrificans DSM 13742 (AF126545) L6B-287 Pseudomonas veronii CIP 104663 (AF064460) 85 Pseudomonas moorei RW10 (AM293566) Pseudomonas umsongensis Ps3-10 (AF468450) 99 L6B-86 48 Pseudomonas thivervalensis CFBP 11261 (AF100323) L6B 161 38 L6B-295 53 L6B-13 L6B-109 73 L6B-148 L6B-302 Pseudomonas frederiksbergensis DSM 13022 (AJ249382) L6B-151 74 L6B-52 64

22

98

93 62

100

Pedobacter steynii DSM 19110 (AM491372) L6B-56 Flavobacterium limicola DSM 15094 (AB075230) 47 L6B-131 59 L6B-23 L6B-222 61 Flavobacterium psychrolimnae LMG 22018 (AJ585428) 100 L6B-17

L6B-127 L6B-150 99 L6B-132 88 Janthinobacterium lividum DSM 1522 (Y08846) 99 L6B-103 99 Janthinobacterium agaricidamnosum DSM 9628 (Y08845) Polaromonas aquatica CCUG 39402 (AM039830) L6B-55 100 Polaromonas naphthalenivorans CJ2 (AY166684) 53 47 L6B-97 Aquifex pyrophilus Kol5a (M83548)

0.02

Fig. 2. Neighbor joining phylogenetic tree of 16S rRNA gene clones from libraries constructed using the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of sediment of Lake 6, Schirmacher Oasis, Antarctica. Clones belonging to the Gram-negative bacteria from all three libraries are included in the tree. Bootstrap values >50% are indicated at the nodes for 1000 replicates. The bar represents 2 substitutions per 100 nucleotides.

PC2-0.028%) explain 99.983% of the total variances. The PCA plot indicated that the bottom sample was slightly different from the subsurface and middle samples, but altogether they were closely associated (Supplementary Fig. 3). 3.8. Comparison of the bacterial community composition determined by T-RFLP, 16S rRNA gene clone libraries and culturable isolates At the phylum/class level of taxonomic discrimination using T-RFLP, 16S rRNA gene clone libraries and culturable isolates, it was possible to detect Beta- and Gamma- proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes (Supplementary Table 2). The other phylum/class such as Alphaproteobacteria, Spirochaetes, Caldiserica and Clostridia were not detectable in the culture method, but could be detected either in the T-RFLP approach and clone library or any of the later approaches (Supplementary Table 2). At the genus level, many more genera were detected in the T-RFLP approach (21 genera) and in clone libraries (17 genera) compared to the culturable isolates (8 genera) (Supplementary Table 2).The increased number of genera detected in the T-RFLP approach is also due to the fact that, at times, the same TRF represents more than one genus and it is not easy to assign the fragment to a single genus. Therefore, in our opinion, the 17 genera detected by clone libraries probably represent the minimum diversity of the sediment. Seven of the genera were common to the T-RFLP approach and clone libraries.

Six out of the 8 genera (except for the genera Microbacterium and Paenisporosarcina) detected as culturable isolates were represented in the T-RFLP approach, indicating that the two methods yield very similar results (Supplementary Table 2). It would also imply that these genera are more amenable to culture but may not be present in large numbers in the soil and therefore were not represented in the clone library. Four of the genera detected in the culturable approach were also detected in the clone library approach and thus it would appear that these four genera, Janthinobacterium, Pseudomonas, Flavobacterium and Paenisporosarcina, respectively, are present in large numbers and are also easily culturable (Supplementary Table 2). 4. Discussion Microorganisms in the sediment play a significant role in remineralization of organic matter within the aquatic ecosystem. In Antarctica, microbial communities have been investigated in continental shelf sediment (Bowman et al., 2003), glacial melt water lake sediment (Sjo¨ling and Cowan, 2003) and Antarctic marine benthos (Reichardt, 1987). In this study, the bacterial community characteristics of Antarctic lake sediment were studied using both culture-dependent and culture-independent approaches (T-RFLP and 16S rRNA gene libraries). The results indicated a low bacterial count and organic carbon percent in the bottom sediment (100e104 cm) compared to the upper depths (18e22 and 60e64 cm). This

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

199

Caldisericum exile AZM16c01 (AB428365)

89 71

Clade 1 (53 clone sequences)

99

91 L6B-389 L6B-381 84

12

Tissierella creatinophila KRE4 (X80227)

99

L6B-432 Clostridium amylolyticum SW408 (EU037903)

68

99 L6B-341 L6B-391

25

Natranaerobius trueperi JW/NM-WN-LU (EU338490)

26

Dethiobacter alkaliphilus AHT1 (ACJM01000032) 99 L6B-413

25

Desulfosporosinus lacus STP12 (AJ582757)

84

86 99

17

L6B-327 Paenisporosarcina macmurdoensis DSM 15428 (AJ514408) L6B-14

99

79

L6B-430 96

L6B-355 L6B-423 L6B-116

57

Catellatospora coxensis 2-29/17 (AB200232)

94 48

76

99

Sporichthya polymorpha IFO 12702 (AB025317) Streptomyces glomeratus LMG 19903 (AJ781754) Streptomyces malaysiensis DSM 41697 (AB249918)

92

L6B-152 Thermobaculum terrenum ATCC BAA-798 (AF391972) L6B-364 Aquifex pyrophilus Kol5a (M83548)

0.02

Fig. 3. Neighbor joining phylogenetic tree of 16S rRNA gene clones from libraries constructed using the subsurface (18e22 cm), middle (60e64 cm) and bottom (100e104 cm) of sediment of Lake 6, Schirmacher Oasis, Antarctica. Clones belonging to the Gram-positive bacteria from all three libraries are included in this tree. The following clones have been compressed: L6B-352, 370, 334, 382, 365, 346, 387, 330, 408, 343, 357, 339, 433, 407, 435, 332, 335, 356, 412, 331, 416, 429, 375, 362, 325, 318, 347, 349, 337, 427, 329, 373, 386, 366, 350, 443, 390, 317, 431, 377, 409, 410, 418, 344, 388, 438, 378, 380, 338, 363, 340, 411, 436 in Clade 1. Bootstrap values >50% are indicated at the nodes for 1,000 replicates. The bar represents 2 substitutions per 100 nucleotides.

may be due to the prevalence of anoxic conditions in this region, which is also supported by clone library data (Supplementary Table 1). The other chemical characteristics, including Li, Be, Na, Mg, Al, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Rb, Sr, Ag, Cd, Te, Ba, Tl, Pb and Bi content, varied slightly at the subsurface, middle and bottom of the sediment sample of lake 6. Except for Cu and Ag content, all the other elements were abundant in the bottom sediment and this may also influence the low bacterial count in the bottom sediment (Table 1). However, PCA based on biogeochemical properties, depth and total bacterial count of the subsurface, middle and bottom of the sediment sample of lake 6 indicated that the three samples were not very different and were closely associated (Supplementary Fig. 3). A large difference was observed in the total bacterial count compared with the CFU, thus confirming earlier observations that only a small fraction of the bacteria are culturable under laboratory conditions. At the same time, it is important to consider the fact that determination of the total bacterial count using the BacLight_Bacterial Viability kit (Invitrogen, Oregon, USA) is a sensitive method but may underestimate the number since, in sediments, the possibility of bacterial cells remaining attached to soil particles cannot be ruled out. In the present study, attempts were made to overcome the problem

by subjecting the sample to shaking for 2 h prior to determining the number. Takii et al. (1986) used acridine orange epifluorescence method and reported bacterial abundance in the bottom water of Vanda lake, Antarctica. It was 107 cells ml1, which is similar to the abundance observed by us in the present study. Using the culturable approach, 41 isolates were characterized; based on phylogenetic analysis, they could be categorized into 14 groups. Considering that all these strains were isolated from the sediment of an Antarctic lake, it was not surprising that the representative strains were either psychrophilic (grow below 20
C) or psychrotolerant (grow, between 4 and 30
C) (Table 3). Taxonomic analysis indicated that species affiliated with the genera Arthrobacter, Cryobacterium, Janthinobacterium and Pseudomonas are the most predominant. Furthermore, 7 of the phylogenetic neighbors of the 14 groups had been isolated earlier from cold habitats and have been reported to be either psychrophilic or psychrotolerant. They include Cryobacterium psychrotolerans (Zhang et al., 2007), Cryobacterium roopkundense (Reddy et al., 2010), Flavobacterium psychrolimnae (Van Trappen et al., 2005), Microbacterium profundii (Wu et al., 2008), Brevibacterium antiquum (Gavrish et al., 2004), Paenisporosarcina macmurdoensis (Krishnamurthi et al., 2009; Reddy et al., 2003) and Janthinobacterium lividum (Shivaji et al., 1991).

200

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

Six of the remaining 7 phylogenetic neighbors were not isolated from the cold habitats, although clones of these species, namely Pseudomonas azotoformans (Srinivas et al., 2009), Arthrobacter oxydans, Arthrobacter scleromae, Arthrobacter sulfonivorans Pseudomonas frederiksbergensis (Vardhan Reddy et al., 2009) and Pseudomonas mandelii (Srinivas et al., 2009; Vardhan Reddy et al., 2009), were reported from different habitats of Arctic and Antarctic regions. However, one species, Pseudomonas gessardii, has never been reported thus far from a cold habitat. Psychrophilic bacteria have attracted the attention of the scientific community (Feller and Gerday, 2003) due to their ability to produce cold active enzymes, with potential applications in a broad range of industrial, agricultural and medical processes. When the enzymatic activities of the isolates (Table 3) were compared with those of their nearest phylogenetic neighbor (Table 2), strains Lc10-2, Lc10-5, Lc10-6, Lc10-7, Lc30-1, Lc302, Lc31-4, Lc31-5, Lc50-1 and Lc51-7 showed properties which did not match their nearest phylogenetic relatives. Strain Lc1-2 exhibited lipase activity and did not exhibit protease activity like the corresponding phylogenetic neighbor Pseudomonas mandelii (Delorme et al., 2002). Strains Lc10-3 and Lc50-3 exhibited both amylase and protease activities, but did not show lipase and urease activities, as in the corresponding phylogenetic neighbors Paenisporosarcina macmurdoensis and Flavobacterium psychrolimnae, respectively (Reddy et al., 2003; Van Trappen et al., 2005). All other strains varied with respect to their ability to exhibit enzymatic activities compared to the nearest phylogenetic neighbor. Though the exact function of these bacteria is difficult to predict, it may be speculated that these bacteria which are isolated from the different sediment depths might play a key role in utilizing the complex organic compounds obtained from the other trophic levels, which is evident from the enzymatic activities exhibited by most of the bacteria at 4
C. Ellis-Evans, (1981a) suggested a similar role for extracellular-enzymeproducing bacteria in sediments of freshwater lakes studied at Signy Island, South Orkney Islands, Antarctica. These bacteria were predominantly proteolytic, with amylolytic and lipolytic bacteria constituting very small components. In the present study, both proteolytic and amylotic bacteria were more predominant than the lipolytic and ureolytic bacteria. Accurate characterization of a microbial community could be achieved by T-RFLP using multiple restriction enzymes (Moeseneder et al., 1999). But in the present study, T-RFLP was mainly used to check variation in the community with depth (Fig. 1) and to tentatively identify bacterial populations within a microbial community (Table 4). At the phylum/class level, TRFs affiliated with Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Spirochaetes (Table 4) were identified. Stratification of bacteria was also observed using the T-RFLP method. For instance, bacteria affiliated with the genus Rhodopseudomonas (TRF of 109 bp) is present only at the subsurface and middle of the sediment, whereas those affiliated with Tissierella (TRF of 467 bp), Polaromonas (TRF of 419 bp) and Cryobacterium (TRF of 453 bp) were absent at the surface and or middle of the sediment, but were definitely present in the bottom portion of the sediment. Those bacteria present in the middle and the bottom part of the sediment are

likely to be anaerobes, like strains affiliated with Rhodopseudomonas and Tissierella. Rhodopseudomonas is a facultative anaerobe and has been earlier reported from Lake Fryxell, McMurdo Dry Valleys, Antarctica (Brambilla et al., 2001). Bacterial distribution in the surface sediments may be complex due to rapid change in physicochemical and geochemical factors along the sediment depth (D’Hondt et al., 2002). Kent et al. (2003) indicated that some phylogenetic groups are not well represented in the TRF database (Kent et al., 2003), but this does not appear to be the case in the present study, since more genera were detected by T-RFLP. Except for the genera Microbacterium and Paenisporosarcina, all 5 remaining genera detected in the culturable isolates were represented in the T-RFLP approach (Supplementary Table 2). Rarefaction analysis indicated that 16S rRNA gene clone libraries were representative of the three sediment layers. Furthermore, the predominant bacteria in the three libraries were affiliated with five major lineages, the Proteobacteria, Bacteroidetes, Actinobacteria, Caldiserica and Firmicutes. Clones affiliated with Alphaproteobacteria were not detected and this was not unexpected, since Alphaproteobacteria are usually regarded as being characteristic of marine environments (Zwisler et al., 2003). The clone libraries also indicated a clear stratification of bacteria with depth. For instance, Gammaproteobacteria, which was dominant at the sediment subsurface (about 70% of the clones) increased and became the most predominant bacteria in the middle of the sediment (about 99% of the clones); it then completely disappeared at the bottom of the sediment, which is now dominated by Caldiserica (about 83% of the clones). Gammaproteobacteria is represented by the genus Pseudomonas and one sequence of Thioalkalivibrio denitrificans. The genus Pseudomonas has been reported from deep-sea sediment samples and also Antarctic mat samples (Shivaji et al., 1989; Kato et al., 1997; Brambilla et al., 2001). The species Thioalkalivibrio denitrificans was reported from sediments of the soda lakes Bogoria and Baer and also from deep-sea sediment in the Japan Trench (Sorokin et al., 2001; Ma et al., 2004; Inagaki et al., 2002). Betaproteobacteria is present only in the surface layer and is represented by two species of the genera Janthinobacterium and Polaromonas which were earlier reported from Lake Fryxell, McMurdo Dry Valleys, Antarctica (Brambilla et al., 2001). The occurrence of Betaproteobacteria in the polar and temperate regions is rare (Brinkmeyer et al., 2003) and this is supported by the occurrence of low frequency distribution of clone sequences in the present study. The clones affiliated with Flavobacteria also decreased with depth, thus confirming an earlier study that had indicated that the incidence of Flavobacterium declined with the depth of the sample and they were practically absent in the 100 cm layer (Bowman and McCuaig, 2003). The clones exhibited similarity with Flavobacterium psychrolimnae, a psychrophilic bacterium isolated from microbial mats in Antarctic lakes (Van Trappen et al., 2005) and Flavobacterium limicola (Tamaki et al., 2003). Many novel members of the family Flavobacteriaceae have been reported from Antarctic habitats (Bowman and Nichols, 2005). One clone showing 98% similarity with the genus Pedobacter steynii was earlier reported from a lake sediment

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

core of Ardley Island, West Antarctica (Li et al., 2006). Thus it appears that the surface layer is mostly occupied by Gammaproteobacteria and Bacteroidetes and this is in accordance with the results of Li et al., (2006). It is possible that Gammaproteobacteria is more tolerant to anoxic conditions and therefore survives even at 60 cm from the surface compared to Betaproteobacteria and Flavobacteria. Furthermore, the absence of Caldiserica in the subsurface and in the middle of the sediment, but its predominance at the bottom of the sediment, would imply that this taxon is anaerobic. In fact, most of the clones (82.8%) in the 100 cm library are related to Caldisericum exile, which is a single representative species of the phylum Caldiserica which is anaerobic, thermophilic and a filamentous bacterium which was originally referred to as the phylum OP5. Clones related to this phylum were earlier reported from different anaerobic habitats like anaerobic thermophilic phenol treatment slurry, permafrost habitat, chlorinated solvent contaminated sediment core and hot spring microbial mat in Iceland (Fang et al., 2006; Liebner et al., 2008; Dojka et al., 1998; Skirnisdottir et al., 2000). However, as yet, there has been no report of isolates of Caldisericum from Antarctica. Clones affiliated with Actinobacteria and Firmicutes were not very dominant (0.8e4.7%) and exhibited similarity with Streptomyces malaysiensis, Streptomyces glomeratus, Catellatospora coxensis and Sporichthya polymorpha. The presence of indigenous Streptomyces populations in marine sediments has been reported (Moran et al., 1995). Clones representing the class Bacilli were affiliated with Paenisporosarcina macmurdoensis which was earlier isolated by us from a pond in the McMurdo Dry valleys, Antarctica (Reddy et al., 2003). Changes in bacterial community structure with depth of the sediment have been previously reported in coastal Pacific (Urakawa et al., 2000), for cold seep sediments (Inagaki et al., 2002) and also in Antarctic (Bowman and McCuaig, 2003; Bowman et al., 2003; Li et al., 2006; Sjo¨ling and Cowan, 2003), where the highest bacterial richness was typically observed in subsurface sediment layers. Such changes have been interpreted as a result of the shift from oxic to anoxic sediment layers. The present study confirms bacterial richness in the subsurface sediment layer and also provides evidence that this may reflect a change in the oxic conditions because, in the deepest part of the sediment at 100e104 cm, the abundance was drastically reduced and predominantly represented by anaerobic bacteria affiliated with the genera Caldisericum, Clostridium, Tissierella, Desulfosporosinus, Natranaerobius and Dethiobacter. Others are facultative anaerobes like Sporichthya (Actinobacteria). Anaerobic bacteria, including facultative anaerobes have been isolated from a variety of Antarctic ecosystems, including lake-water column and sediments (Franzmann and Rohde, 1991). Out of the 6 clones of class Clostridia, one clone showing similarity with Clostridium amylolyticum is a strict anaerobic bacterium. Clostridium genera have been earlier reported from lake sediments of Schirmacher Oasis, Antarctica (Alam et al., 2006). Thermoanaerobacter, which was detected using only T-RFLP approach, was earlier reported from marine salinity meromictic lakes and

201

a coastal meromictic marine basin, Vestfold Hills, Eastern Antarctica by Bowman et al., (2000). It was anticipated that the stratification of a bacterial community observed in this study would be similar to that observed earlier in a 59 cm lake sediment core of Ardley Island, West Antarctica (Li et al., 2006). Indeed, there were many similarities, for instance, Gammaproteobacteria, Gemmatimonadetes, CFB, Firmicutes and Actinobacteria were common in the surface up to 64 cm in both studies. In addition, in the 59 cm lake sediment core of Ardley Island, West Antarctica (Li et al., 2006), putative sulfur- or sulfideoxidizing Deltaproteobacteria were detected in the middle layers (Li et al., 2006) which were totally unrepresented in the present study. Thus, it would appear that similar niches are also not very similar in Antarctica and this may represent intrinsic differences in the physicochemical properties of the lakes which need to be studied in greater detail. That the variation in the bacterial community is dependent on the specific niche under study is also obvious from the fact that Bowman et al. (2003) observed that in Antarctic continental shelf sediments, the microbial community structure was homogenous in the sediment core depths of 1e4 cm and was represented by Gamma- and Delta- proteobacteria, Flavobacteria, Planctomycetales and Archaea. Furthermore, the bacterial community in glacial melt water lake sediment in Antarctica was represented by Alpha-, Gamma- and Delta- proteobacteria, the CFB group, the Spirochaetacea and the Actinobacteria (Sjo¨ling and Cowan, 2003). The above studies do indicate that certain bacteria like Gammaproteobacteria, CFB, Firmicutes and Actinobacteria are dominant in the sediments of Antarctica. In conclusion, this study on the bacterial diversity of a sediment sample at the subsurface, middle and bottom of a sediment core sampled from a freshwater lake in Antarctica confirms that bacterial richness in the subsurface sediment layer is greater than in deeper regions of the sediment; it provides evidence that anaerobic bacteria are predominant in the deep anoxic parts of the sediment and variations in the bacterial community are dependent on the specific niche that is being studied. Based on comparison with earlier studies, the present work also suggests that bacteria affiliated with Gammaproteobacteria, CFB, Firmicutes and Actinobacteria are dominant in the sediments of Antarctica. Acknowledgements We would like to thank the National Center for Antarctic and Ocean Research, Goa and the CSIR Network Project titled “Exploitation of India’s rich microbial diversity” (NWP0006) for funding. TNRS acknowledges the CSIR, the Government of India for the award of a Research Associate position.

Appendix. Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.resmic.2010.09.020.

202

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203

References Alam, S.I., Dixit, A., Reddy, G.S.N., Dube, S., Palit, M., Shivaji, S., Singh, L., 2006. Clostridium schirmacherense sp. nov., an obligately anaerobic, proteolytic, psychrophilic bacterium isolated from lake sediment of Schirmacher Oasis, Antarctica. Int. J. Syst. Evol. Microbiol. 56, 715e720. Altmann, D., Stief, P., Amann, R., De Beer, D., Schramm, A., 2003. In situ distribution and activity of nitrifying bacteria in freshwater sediment. Environ. Microbiol. 5, 798e803. Bowman, J.P., McCuaig, R.D., 2003. Biodiversity, community structural shifts and biogeography of prokaryotes within Antarctic continental shelf sediment. Appl. Environ. Microbiol. 69, 2463e2483. Bowman, J.P., Nichols, D.S., 2005. Novel members of the family Flavobacteriaceae from Antarctic maritime habitats including Subsaximicrobium wynnwilliamsii gen. nov., sp. nov., Subsaximicrobium saxinquilinus sp. nov., Subsaxibacter broadyi gen. nov., sp. nov., Lacinutrix copepodicola gen. nov., sp. nov., and novel species of the genera Bizionia, Gelidibacter and Gillisia. Int. J. Syst. Evol. Microbiol. 55, 1471e1486. Bowman, J.P., Rea, S.M., McCammon, S.A., McMeekin, T.A., 2000. Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hills, Eastern Antarctica. Environ. Microbiol. 2, 227e237. Bowman, J.P., McCammon, S.A., Gibson, J.A.E., Robertson, L., Nichols, P.D., 2003. Prokaryotic metabolic activity and community structure in Antarctic continental shelf sediment. Appl. Environ. Microbiol. 69, 2448e2462. Brambilla, E., Hippe, H., Hagelstein, A., Tindall, B.J., Stackebrandt, E., 2001. 16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica. Extremophiles 5, 23e33. Brinkmeyer, R., Knittel, K., Jurgens, J., Weyland, H., Amann, R., Helmke, E., 2003. Diversity and structure of bacterial communities in Arctic versus Antarctic pack ice. Appl. Environ. Microbiol. 69, 6610e6619. Costello, A.M., Auman, A.J., Macalady, J.L., Scow, K.M., Lidstrom, M.E., 2002. Estimation of methanotroph abundance in a freshwater lake sediment. Environ. Microbiol. 4, 443e450. Covert, J.S., Moran, M.A., 2001. Molecular characterization of estuarine bacterial communities that use high- and low-molecular weight fractions of dissolved organic carbon. Aquat. Microbiol. Ecol. 25, 127e139. D’Hondt, S., Rutherford, S., Spivack, A.J., 2002. Metabolic activity of subsurface life in deep-sea sediments. Science 295, 2067e2070. Delorme, S., Lemanceau, P., Christen, R., Corberand, T., Meyer, J.M., Gardan, L., 2002. Pseudomonas lini sp. nov., a novel species from bulk and rhizospheric soils. Int. J. Syst. Evol. Microbiol. 52, 513e523. Dojka, M.A., Hugenholtz, P., Haack, S.K., Pace, N.R., 1998. Microbial diversity in a hydrocarbon and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl. Environ. Microbiol. 64, 3869e3877. Ellis-Evans, J.C., 1981a. Freshwater Microbiology at Signy Island, South Orkney Islands. Antarctica. PhD. CNAA, 283 pp. Ellis-Evans, J.C., 1996. Microbial diversity and function in Antarctic freshwater ecosystems. Biodiversity and Conservation 5, 1395e1431. Fang, H.H., Liang, D.W., Zhang, T., Liu, Y., 2006. Anaerobic treatment of phenol in wastewater under thermophilic condition. Water Res. 40, 427e434. Feller, G., Gerday, C., 2003. Psychrophilic enzymes: hot topics in cold adaptation. Nat. Rev. Microbiol. 1, 200e208. Franzmann, P.D., Rohde, M., 1991. An obligately anaerobic, coiled bacterium from Ace Lake, Antarctica. J. Gen. Microbiol. 137, 2191e2196. Gavrish, E.Y., Krauzova, V.I., Potekhina, N.V., Karasev, S.G., Plotnikova, E.G., Altyntseva, O.V., Korosteleva, L.A., Evtushenko, L.I., 2004. Three new species of Brevibacteria, Brevibacterium antiquum sp. nov., Brevibacterium aurantiacum sp. nov., and Brevibacterium permense sp. nov. Mikrobiologiya 73, 176e183. Gregory, L.G., Bond, P.L., Richardson, D.J., Spiro, S., 2003. Characterization of a nitrate-respiring bacterial community using the nitrate reductase gene (narG) as a functional marker. Microbiology 149, 229e237. Inagaki, F., Sakihama, Y., Inoue, A., Kato, C., Horikoshi, K., 2002. Molecular phylogenetic analyses of reverse-transcribed bacterial rRNA obtained from deep-sea cold seep sediments. Environ. Microbiol. 4, 277e286.

Junier, P., Junier, T., Witzel, K.P., 2008. TRiFLe, a program for in silico terminal restriction fragment length polymorphism analysis with userdefined sequences sets. Appl. Environ. Microbiol. 74, 6452e6456. Jurgens, G., Glo¨ckner, F., Amann, R., Saano, A., Montonen, L., Likolammi, M., Mu¨nster, U., 2000. Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization. FEMS Microbiol. Ecol. 34, 45e56. Kato, C., Li, L., Tamaoka, J., Horikoshi, K., 1997. Molecular analyses of the sediment of the 11000-m deep Mariana Trench. Extremophiles 1, 117e123. Kent, A.D., Smith, D.J., Benson, B.J., Triplett, E.W., 2003. Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities. Appl. Environ. Microbiol. 69, 6768e6776. Krishnamurthi, S., Bhattacharya, A., Mayilraj, S., Saha, P., Schumann, P., Chakrabarti, T., 2009. Description of Paenisporosarcina quisquiliarum gen. nov., sp. nov., and reclassification of Sporosarcina macmurdoensis Reddy et al., 2003 as Paenisporosarcina macmurdoensis comb. nov. Int. J. Syst. Evol. Microbiol. 59, 1364e1370. Li, S., Xiao, X., Yin, X., Wang, F., 2006. Bacterial community along a historic lake sediment core of Ardley Island, west Antarctica. Extremophiles 10, 461e467. Li, F.H., Meredith, A.J., Lampe, J.W., 2007. Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota. J. Microbiol. Meth. 68, 303e311. Liebner, S., Harder, J., Wagner, D., 2008. Bacterial diversity and community structure in polygonal tundra soils from Samoylov Island, Lena Delta Siberia. Int. Microbiol. 11, 195e202. Luna, G.M., Anno, D.A., Giuliano, L., Danovaro, R., 2004. Bacterial diversity in deep Mediterranean sediments: relationship with the active bacterial fraction and substrate availability. Environ. Microbiol. 6, 745e753. Ma, Y., Zhang, W., Xue, Y., Zhou, P., Ventosa, A., Grant, W.D., 2004. Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses. Extremophiles 8, 45e51. Moeseneder, M.M., Arrieta, J.M., Muyzer, G., Winter, C., Herndl, G.J., 1999. Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 65, 3518e3525. Moran, M.A., Rutherford, L.T., Hodson, R.E., 1995. Evidence for indigenous Streptomyces populations in a marine environment determined with a 16S rRNA probe. Appl. Environ. Microbiol. 61, 3695e3700. Mummey, D.L., Stahl, P.D., 2003. Spatial and temporal variability of bacterial 16S rDNA-based T-RFLP patterns derived from soil of two Wyoming grassland ecosystems. FEMS Microbiol Ecol. 46, 113e120. Nakano, Y., Takeshita, T., Kamio, N., Shiota, S., Shibata, Y., Yasui, M., Yamashita, Y., 2008. Development and application of a T-RFLP data analysis method using correlation coefficient matrices. J. Microbiol. Meth. 75, 501e505. Pradhan, S., Srinivas, T.N., Pindi, P.K., Kishore, K.H., Begum, Z., Singh, P.K., Singh, A.K., Pratibha, M.S., Yasala, A.K., Reddy, G.S., Shivaji, S., 2010. Bacterial biodiversity from Roopkund glacier, Himalayan mountain ranges, India. Extremophiles 14, 377e395. Purdy, K.J., Nedwell, D.B., Embley, T.M., 2003. Analysis of the sulfatereducing bacterial and methanogenic archaeal populations in contrasting Antarctic sediments. Appl. Environ. Microbiol. 69, 3181e3191. Vardhan Reddy, V.P., Nageswara Rao, S.S.S., Pratibha, M.S., Sailaja, B., Kavya, B., Ruth Manorama, R., Singh, S.M., Srinivas, T.N.R., Shivaji, S., 2009. Bacterial diversity and bioprospecting for cold-active enzymes from culturable bacteria associated with sediment of melt water stream of Midtre Lov’enbreen glacier, an Arctic glacier. Res. Microbiol. 160, 538e546. Reddy, G.S.N., Matsumuto, G.I., Shivaji, S., 2003. Sporosarcina macmurdoensis sp. nov., from a cyanobacterial mat sample from a pond in the McMurdo Dry Valleys, Antarctica. Int. J. Syst. Evol. Microbiol. 53, 1363e1367. Reddy, G.S.N., Pradhan, S., Manorama, R., Shivaji, S., 2010. Cryobacterium roopkundense sp. nov., a psychrophilic bacterium isolated from glacial soil. Int. J. Syst. Evol. Microbiol. 60, 866e870.

S. Shivaji et al. / Research in Microbiology 162 (2011) 191e203 Reichardt, W., 1987. Differential temperature effects on the efficiency of carbon pathways in Antarctic marine benthos. Mar. Ecol. Prog. Ser. 40, 127e135. Schu¨tte, U.M., Abdo, Z., Bent, S.J., Shyu, C., Williams, C.J., Pierson, J.D., Forney, L.J., 2008. Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities. Appl. Microbiol. Biotechnol. 80, 365e380. Shivaji, S., Rao, N.S., Saisree, L., Sheth, V., Reddy, G.S.N., Bhargawa, M., 1989. Isolation and identification of Pseudomonas species from Schirmacher oasis, Antarctica. Appl. Environ. Microbiol. 55, 767e771. Shivaji, S., Ray, M.K., Seshu, K.G., Reddy, G.S.N., Saisree, L., WynnWilliams, D.D., 1991. Identification of Janthinobacterium lividum from the soils of the islands of Scotia Ridge and from Antarctic peninsula. Pol. Biol. 11, 267e271. Shivaji, S., Ray, M.K., Rao, N.S., Saisree, L., Jagannadham, M.V., Kumar, G. S., Reddy, G.S.N., Bhargava, P.M., 1992. Sphingobacterium antarcticus sp. nov., a psychrotrophic bacterium from the soils of Schirmacher Oasis, Antarctica. Int. J. Syst. Bacteriol. 42, 102e106. Shivaji, S., Chaturvedi, P., Begum, Z., Pindi, P.K., Manorama, R., Padmanaban, D.A., Shouche, Y.S., Pawar, S., Vaishampayan, P., Dutt, C.B. , Datta, G.N., Manchanda, R.K., Rao, U.R., Bhargava, P.M., Narlikar, J.V., 2009. Janibacter hoylei sp. nov., Bacillus isronensis sp. nov. and Bacillus aryabhattai sp. nov., isolated from cryotubes used for collecting air from the upper atmosphere. Int. J. Syst. Evol. Microbiol. 59, 2977e2986. Shyu, C., Soule, T., Bent, S.J., Foster, J.A., Forney, L.J., 2007. MiCA: a webbased tool for the analysis of microbial communities based on terminalrestriction fragment length polymorphisms of 16S and 18S rRNA genes. J. Microbiol. Ecol. 53, 562e570. Sjo¨ling, S., Cowan, D.A., 2003. High 16S rDNA bacterial diversity in glacial meltwater lake sediment, Bratina Island, Antarctica. Extremophiles 7, 275e282. Skirnisdottir, S., Hreggvidsson, G.O., Hjorleifsdottir, S., Marteinsson, V.T., Petursdottir, S.K., Holst, O., Kristjansson, J.K., 2000. Influence of sulfide and temperature on species composition and community structure of hot spring microbial mats. Appl. Environ. Microbiol. 66, 2835e2841. Sorokin, D.Y., Lysenko, A.M., Mityushina, L.L., Tourova, T.P., Jones, B.E., Rainey, F.A., Robertson, L.A., Kuenen, G.J., 2001. Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis

203

sp.nov., novel and Thioalkalivibrio denitrificancs sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes. Int. J. Syst. Evol. Microbiol. 51, 565e580. 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. Takii, S., Konda, T., Hiraishi, A., Matsumoto, G.I., Kawano, T., Torii, T., 1986. Vertical distribution in and isolation of bacteria from Lake Vanda: an Antactic lake. Hydrobiologia 135, 15e21. Tamaki, H., Hanada, S., Kamagata, Y., Nakamura, K., Nomura, N., Nakano, K. , Matsumura, M., 2003. Flavobacterium limicola sp. nov., a psychrophilic, organic-polymer-degrading bacterium isolated from freshwater sediments. Int. J. Syst. Evol. Microbiol. 53, 519e526. Urakawa, H., Yoshida, T., Nishimura, M., Ohwada, K., 2000. Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA based approaches and quinone profiling. Environ. Microbiol. 2, 542e554. Van Trappen, S., Vandecandelaere, I., Mergaert, J., Swings, J., 2005. Flavobacterium fryxellicola sp. nov. and Flavobacterium psychrolimnae sp. nov., novel psychrophilic bacteria isolated from microbial mats in Antarctic lakes. Int. J. Syst. Evol. Microbiol. 55, 769e772. Wu, Y.H., Wu, M., Wang, C.S., Wang, X.G., Yang, J.Y., Oren, A., Xu, X.W., 2008. Microbacterium profundii sp. nov., isolated from deep-sea sediment of polymetallic nodule environments. Int. J. Syst. Evol. Microbiol. 58, 2930e2934. Yeates, C., Gillings, M.R., Davison, A.D., Altavilla, N.M., Veal, D.A., 1998. Methods for microbial DNA extraction from soil for PCR amplification. Biol. Procedures. Online 1, 40e47. Zhang, D.C., Wang, H.X., Cui, H.L., Yang, Y., Liu, H.C., Dong, X.Z., Zhou, P. J., 2007. Cryobacterium psychrotolerans sp. nov., a novel psychrotolerant bacterium isolated from the China No. 1 glacier. Int. J. Syst. Evol. Microbiol. 57, 866e869. Zwisler, W., Selje, N., Simon, M., 2003. Seasonal patterns of the bacterioplankton community composition in a large mesotrophic lake. Aquatic. Microbiol. Ecol. 31, 211e225.