Phylogenetic profiling of bacterial community from two intimately located sites in Balramgari, North-East coast of India.

Abstract

Microbial communities in coastal subsurface sediments play an important role in biogeochemical cycles. In this study microbial communities in tidal subsurface sediments of Balramgari in the state of Orissa, India were investigated using a culture independent approach. Two 16S rDNA cloned libraries were prepared from the closely located (100 m along the coast) subsurface sediment samples. Library I sediment samples had higher organic carbon content but lower sand percentage in comparison to Library II. A total of 310 clone sequences were used for DOTUR analysis which revealed 51 unique phylotypes or operational taxonomic units (OTUs) for both libraries. The OTUs were affiliated with 13 major lineages of domain bacteria including Proteobacteria (α, β, δ and λ), Acidobacteria, Actinobacteria, Cyanobacteria, Chloroflexi, Firmicutes, Verrucomicrobia, Bacteroidetes, Gemmatimonadetes and TM7. We encountered few pathogenic bacteria such as Aeromonas hydrophila and Ochrobactrum intermedium, in sediment from Library I. ∫-LIBSHUFF comparison depicts that the two libraries were significantly different communities. Most of the OTUs from both libraries possessed ≥85% to <97% similarity to RDP database sequences depicting the putative presence of new species, genera and phylum. This work revealed the complex and unique bacterial diversity from coastal habitat of Balramgari and shows that, in coastal habitat a variability of physical and chemical parameter has a prominent impact on the microbial community structure.

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Indian J Microbiol (June 2009) 49:169–187
Indian J Microbiol (June 2009) 49:169–187
169
ORIGINAL ARTICLE
Phylogenetic profi ling of bacterial community from two
intimately located sites in Balramgari, North-East coast of
India
Arvind Kumar Gupta · Ashraf Yusuf Rangrez · Pankaj Verma · Anil Chatterji · Yogesh S. Shouche
Received: 13 October 2008 / Accepted: 9 January 2009
DOI: 10.1007/s12088-009-0034-9
Abstract Microbial communities in coastal subsurface
sediments play an important role in biogeochemical cycles.
In this study microbial communities in tidal subsurface
sediments of Balramgari in the state of Orissa, India were
investigated using a culture independent approach. Two
16S rDNA cloned libraries were prepared from the closely
located (100 m along the coast) subsurface sediment sam-
ples. Library I sediment samples had higher organic carbon
content but lower sand percentage in comparison to Library
II. A total of 310 clone sequences were used for DOTUR
analysis which revealed 51 unique phylotypes or opera-
tional taxonomic units (OTUs) for both libraries. The OTUs
were affi liated with 13 major lineages of domain bacteria
including Proteobacteria (α, β, δ and γ), Acidobacteria,
Actinobacteria, Cyanobacteria, Chlorofl exi, Firmicutes,
Verrucomicrobia, Bacteroidetes, Gemmatimonadetes and
TM7. We encountered few pathogenic bacteria such as
Aeromonas hydrophila and Ochrobactrum intermedium, in
sediment from Library I. ∫-LIBSHUFF comparison depicts
that the two libraries were signifi cantly different communi-
ties. Most of the OTUs from both libraries possessed ≥85%
to <97% similarity to RDP database sequences depicting
the putative presence of new species, genera and phylum.
This work revealed the complex and unique bacterial di-
versity from coastal habitat of Balramgari and shows that,
in coastal habitat a variability of physical and chemical
parameter has a prominent impact on the microbial com-
munity structure.
Keywords Ecosystem · Microbial diversity · Marine
sediment · 16S rDNA
Introduction
For millions of years after emergence of the fi rst life forms,
microbial life in the oceans infl uenced the planet’s chemis-
try, altering the chemical balance of the oceans and atmo-
sphere and introducing gradients of oxidizing and reducing
agents. Marine microbial life thrives not only in the sur-
face waters, but also in the lower and abyssal depths from
coastal to the offshore regions, and from the general oceanic
to the specialized niches such as blue waters of coral reefs
to black smokers of hot thermal vents at the sea fl oor [1].
These microbes harbor a unique metabolic machinery to
carry out many steps of the biogeochemical cycles, the
smooth functioning of these cycles is necessary for life to
continue on earth.
Our perspective on microorganisms in the environment
has depended in the past primarily on studies of pure cul-
tures in the laboratory. This view of microbial diversity is
limited as it is estimated that more than 99% of organisms
seen microscopically are not cultivated by routine tech-
niques [2]. Now with the advent of sequence-based taxo-
nomic framework of molecular phylogeny, one requires
A. K. Gupta1 · A. Y. Rangrez1 · P. Verma1 · A. Chatterji2 ·
Y. S. Shouche1 (?)
1Molecular Biology Unit,
National Centre for Cell Science,
Pune - 411 007, Maharashtra, India
2Institute of Tropical Aquaculture,
University Malaysia Terengganu, Malaysia
E-mail: yogesh@nccs.res.in

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170 Indian J Microbiol (June 2009) 49:169–187
123
the gene sequences for identifying the types of organism
occurring in any microbial community. Functioning and
health of an ecosystem is determined to be contingent on its
biodiversity and community structure. Thus, it is important
to study the occurrence of prokaryotic phylotypes and their
distributions in natural communities, which can be carried
out by sequencing 16S rRNA genes obtained from DNA
isolated directly from the environment [3–5].
The northern part of the Bay of Bengal enjoys a tropical
climate, governed mainly by the monsoons. The Southwest
monsoon brings rainfall to the coastal areas from June to
October. The temperature oscillation between summer and
winter is less than 5°C. In contrast to the small variations
in the temperature, the salinity showed wide fl uctuations
(22.4% to 33.4%). This was largely due to the riverine
fl ow from two major river systems, the Ganges and Brah-
maputra, and a number of smaller rivers such as the Subar-
narekha, Budhabalang, Dhamra and Mahanadi [6]. A large
number of Indian horseshoe crabs have been reported to
migrate towards the sandy shore of Balramgari, a unique
beach along north-east coast of India, for breeding purpose
throughout the year. The geo-morphological and eco-bio-
logical characteristics of this beach are totally different with
respect to any other beaches of India [7]. These features
make this environment unique and directs toward exploring
the environmentally important diversity.
The aim of this work was to construct and analyse the
16S rDNA clone libraries to elucidate the genetic diversity
and differences of microbial communities in subsurface
sediments of Balramgari, North-East coast of India.
Materials and methods
Sample collection
Sediment samples utilized for the construction of libraries
were collected from two points (for library I and II) from the
coast of Balramgari, Orissa (Lat 19°16′ N; Long 84°53′ E),
India (Fig. 1). The collection of the sediment was done in
coincidence with the lowest low tide at 1031 hours (tide
height ~0.31 m) on 23 July 2005. Four subpoints at a dis-
tance of 5 m away from each point at midtide level were
selected for collecting the sediment samples. Sediment
Fig. 1 Map showing the place of sample collection, Balramgari, India (Lat 19°16′ N; Long 84°53′ E)

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Indian J Microbiol (June 2009) 49:169–187 171
samples were collected by using a PVC core (depth 5 cm
and diameter 5 cm).
Sediment samples for Library I (L-I) construction were
collected ~100 m away along the cost towards south from
sediment samples collected for Library II (L-II). Samples
were immediately transported in sterile bottles in dry ice
and stored at –80°C until analysis. The temperature and
pH at each sampling site were measured using a centigrade
thermometer and a portable pH meter (Philips 4012). The
sediment samples were analyzed for their grain size dis-
tribution and organic carbon content [8]. The water con-
tent of the sediment was estimated immediately after the
collection by drying a known weight of it at 100oC for 3
hours fol lowed by the re-weighing of the sample. The loss
in weight was ex pressed as a percentage of water content
of the sediment. The organic content of the sediment was
estimated using the chromic acid oxidation method [9]. The
description of physicochemical characteristics of sediments
are summarized in Table 1.
Extraction of total community DNA from sediments
Sediment cores were sliced and upper 0–1 cm layer was re-
moved. Sediments from 1–3 cm sections were mixed and 1 g
of the mixture was used for DNA extraction. Total community
DNA was extracted from all eight samples using MoBio soil
DNA isolation kit (Imperial Bio-Medic) as per the protocol
provided by the manufacturer and the integrity of the extract-
ed DNA was checked by 0.8% horizontal agarose gel elec-
trophoresis and ethidium bromide staining. The concentration
of extracted DNA was checked with the help of NanoDrop
ND-1000 Spectrophotometer (NanoDrop Biotechnologies,
USA) and was ranged between 200 and 300 ng/ μl. DNA from
replicate samples was pooled and stored at –20°C.
PCR and cloning of 16S rRNA gene
16S rRNA gene amplifi cation was performed from both
the pooled extracts in triplicates using universal eubacte-
rial primer set 530F (5’ GTC CCA GCM GCC GCG G 3’)
and 1490R (5’ GGT TAC CTT GTT ACG ACT T 3’) [10].
Amplifi cation was carried out in a 25 μl reaction mixture
containing 200 μM (each) dNTPs, 25 pM each primer, 1 μl
(~50 ng) DNA template and 2.5 U of Taq DNA polymerase
(Bangalore Genei, Bangalore, India) with 1X reaction buf-
fer supplied by the manufacturer. The reaction mixtures
were incubated in GeneAmp PCR System 9700 (Applied
Biosystems) at 94°C for 5 min (initial denaturation and ac-
tivation of Taq DNA polymerase), followed by 25 cycles at
94°C for 1 min (denaturation), 55°C for 1 min (annealing),
and 72°C for 1 min (extension), followed by fi nal extension
for 10 min at 72°C. A tube without DNA was taken as nega-
tive control. Amplifi cation was done only for 25 cycles to
minimize the bias in PCR amplifi cation. 2 μl of amplifi ed
DNA was examined by horizontal electrophoresis on 1%
agarose gel in TAE buffer (40 mM Tris, 20 mM acetate,
2mM EDTA). To minimize the PCR drift, all the three am-
plifi ed PCR products from each site were pooled and puri-
fi ed with Qiagen PCR purifi cation kit (Qiagen, USA) as per
manufacturers’ instruction.
16S rRNA gene library construction and sequencing
Eubacterial libraries were constructed from pooled and
purifi ed PCR product by cloning into pGEM-T easy vector
(Promega, USA) according to manufacturer’s instructions.
Transformation was done in chemically competent Esch-
erichia coli JM109 cells (Stratagen) with 30 minutes recov-
ery time. Positive colonies from the library were picked out
and screened by colony PCR using vector specifi c primers.
The amplifi cation reaction described as above was used and
the PCR conditions were as follows: incubation for 7 min
at 94°C (mainly for cell rupture), 35 cycles each of 1 min
at 94°C (denaturation), at 55°C (primer annealing) and at
72°C (primer extension) and a fi nal elongation step for 10
min at 72°C. A total of 323 clones with proper insert (~960
bp) were purifi ed and sequenced in both directions with an
automated ABI-PRISM 3730 system (Applied BioSystems
Inc.).
Table 1 Analysis of physicochemical parameters of the sediment samples
No.ParametersLibrary I sediment Library II sediment
1Sediment temperature30.5°C 31.6°C
2Sediment pH7.5 7.6
3 Salinity 16.0 x 10-3
17.2 x 10-3
4Organic carbon content2.12 mg c/g0.46 mg c/g
5 Sand percentage 78.32% 83.47%
6 Silt and clay21.68% 16.53%
7 Percentage of water content 5.0% 5.9%
8 Mean grain size0.129 mm0.231 mm

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172 Indian J Microbiol (June 2009) 49:169–187
123
Real-time PCR
Qualitative PCR was performed using 7300 Real Time
PCR System (Applied Biosystems) and Power SYBR
Green PCR master mix supplied by Applied Biosystems.
Amplifi cation was carried out in a 25 μl reaction mixture
containing 50 ng of DNA, 25 pM each primer and Power
SYBR Green PCR master mix. The conditions for the am-
plifi cation of ascV gene (Aeromonas specifi c gene) (ascV-F
5’ TAA RCA GAT GAG TAT CGA TGG 3’ and ascV-R 5’
GAG ACS CGG GTG ACG ATA AT 3’), and recA gene
(Ochrobactrum specifi c gene) (RecA_F 5’ GCG CCG AAA
TCG AAG GT 3’ and RecA_R 5’ GCG AAC CGA ACA
TCA CAC C 3’) and 16S rRNA gene (as an internal con-
trol), were as follows: One denaturation step at 95ºC for 10
min, 40 cycles consisting of denaturation at 95ºC for 15 sec,
a common step for annealing and elongation at 60ºC for 60
sec. At the end of the PCR, the samples were subjected to
melting curve analysis.
Taxonomic and phylogenetic assessment
The partial DNA sequences obtained with the vector
specifi c primers M13F and M13R were assembled and
edited with ChromasPro version 1.33 software. (www.
technelysium.com.au/ChromasPro.html). Vector sequences
were removed from both the ends. Good quality sequences
of approximately 900 nucleotides were used for subse-
quent analysis. Both libraries were analysed for chimeric
sequences and other anomalies using MALLARD software
[11] by using pair-wise comparisons within a multiple
alignment. Each putative chimera identifi ed by the program
was checked with BLASTn [12] and further compared with
closest cultured similar rDNA sequences retrieved from
the DNA databases. Suspected chimeras were excluded
from further analyses. Multiple sequence alignments were
performed using ClustalW version 1.8 [13]. Multiple se-
quence alignment was edited and corrected manually using
DAMBE [14] to get unambiguous sequence alignment.
Appropriate subsets of 16S rDNA sequences were selected
on the basis of initial results and subjected to further phy-
logenetic analysis using the neighbour-joining method
implemented through DNADIST from the PHYLIP version
3.61 [15]. OTUs were generated using furthest neighbour
algorithm of DOTUR program [16]. OTUs generated at
0.03 E.D. (Evolutionary Distance) or the OTUs formed
by the sequences that present a similarity equal or greater
than 97% [17] were used for taxonomic assessment. One
representative clone sequence from each OTU was taken
for taxonomic assessment and it was carried out using se-
quence match programme of RDP (www.rdp.cme.msu.edu/
seqmatch/ seqmatch_intro.jsp). Phylogenetic analysis was
carried out using bayesian inference method with pro-
gram MrBayes 3.0 [18]. The analysis for 16S rRNA gene
consisted of 3,000,000 generations. An appropriate model
of sequence evolution for data set was chosen via Akaike
Information Criterion (AIC) using program MrModeltest
2.2. The model selected was (GTR+I+G) for both the data
sets. Trees were sampled for every 100 generations. First
3000 trees (10%) were discarded as burnin. Bayesian pos-
terior probabilities were calculated using 50% majority rule
consensus and three independent runs were performed for
each dataset. Representative sequences have been assigned
GenBank accession numbers EF451854-EF451955 and
EU236787-EU236933.
Statistical comparison of clone libraries
Good’s coverage [19] was calculated using formula, [1-(n/
N)] X 100 (where n is the number of single clone OTUs and
N is the library size). Rarefaction curves were drawn using
the algorithm described by Hurlbert [20] and plotted as the
number of OTUs versus the number of clones, assuming
that one OTU is formed by the sequences that present a
similarity equal or greater than 97%. Two libraries gener-
ated were compared using ∫-LIBSHUFF program version
1.21 [21] which uses a Monte Carlo procedure to calcu-
late the probability that the observed difference between
two libraries are due to chance. Biodiversity indices were
determined at E.D. 0.03 or a sequence similarity value of
97% for bacterial population using the Shannon Index (H’ =
-Upi*lnpi) which was used as a measure of diversity includ-
ing richness and evenness, the Simpson Dominance Index
[SI’ = n (n-1)/N (N-1)] [22] and the ChaoI estimator as an
alternative to Shannon diversity.
Results
Analysis of 16S rDNA clone libraries
Two hundred white colonies each from both the libraries
were randomly picked and amplifi ed, out of which, 323
positive clones having around 960 bp 16S rDNA insert were
sequenced in both direction using vector specifi c M13F and
M13R primers. Among the 323 rDNA sequences from the
sediment analyzed, we have found 9 of 171 sequences from
L-II to deviate by more than 5% to the E. coli K-12 16S
rDNA sequence taken as standard [23] in the MALLARD
programme, so considered as chimeras. We found only 4
chimeras out of 152 sequences from L-I. All chimeric se-
quences found, can be split into two fragments which show
a reasonably high degree of homology to two very different

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Indian J Microbiol (June 2009) 49:169–187 173
bacteria, however none of the other sequences showed this
phenomenon. These chimeras were neither submitted to
database nor included for further analyses. Thus, chimeras
obtained in this study were much less than those reported
by Wang and Wang [24]. We found that increasing the rep-
licates (three replicates in this study) for PCR and reducing
the reaction cycles to 25 was helpful in reducing PCR drift.
Finally we had 148 clones from L-I and 162 clones from
L-II for further analysis. Using DOTUR programme we ob-
tained 51 OTUs each from both libraries at 0.03 evolution-
ary distance. Each OTU represent a phylotypes and may be
representative of a bacterial species.
Taxonomic distribution of 16S rDNA clone
The distribution of clones in each OTU, their closest cul-
tured and uncultured homologs with similarity score to se-
quence match of RDP database for both library were given
in detail in Tables 2 and 3. The clones obtained were distrib-
uted in various groups of bacteria such as Proteobacteria
(α, β, δ and γ), Acidobacteria, Actinobacteria, Cyanobac-
teria, Chlorofl exi, Firmicutes, Gemmatimonadetes, Bac-
teroidetes, Verrucomicrobia and Candidate divison TM7
(Fig. 2). Proteobacteria was the dominant class of bacteria
identifi ed in both the libraries as also been represented in
other studies. L-I was composed of 80.38% whereas L-II
contained 88.88% of the total clones of Proteobacteria,
unevenly distributed within four subdivisions α, β, γ and δ.
Among Proteobacteria, γ-proteobacteria was the dominant
subdivision in both the libraries with 59.44% clones in L-I
and 67.67% in L-II. The second most abundant subdivision
was α-proteobacteria with 16.21% clones in L-I while δ-
proteobacteria with 11.11% clones in L-II. β-proteobac-
teria were least represented with around 0.67% and 3.7%
clones in L-I and L-II respectively. The second dominant
taxon in L-I was the high G + C gram-positive bacteria
(Actinobacteria) with 6.08% clone, while in L-II, it was
Acidobacteria with 4.3% clones. All the clones affi liated in
the class of Actinobacteria, Acidobacteria, Bacteroidetes,
Gemmatimonadetes, Candidate division TM7 and unclassi-
fi ed bacteria showed homology with uncultured bacterium
of soil and sediments with similarity score between 89 and
98% and did not match with any closest cultured relative
(Tables 2 and 3). Although, both libraries share common
lineages, certain differences were observed, Gemmati-
monadetes (2.02% clones), Fermicutes (2.7% clones) and
Candidate divison TM7 (1.35% clones) were present in L-I
but absent in L-II whereas Cyanobacteria (1.23% clones)
and Chlorofl exi (0.61% clones) were present only in L-II
(Fig. 2).
Phylogenetic reconstruction of 16S rDNA clones
Phylogenetic reconstruction was carried out using Bayesian
inference to determine the relationship of these OTUs to
known sequences of database. Phylogenetic reconstruction
revealed that all OTUs were clustering within 13 major
clades belonging to different division of bacteria. Phyloge-
netic clustering of all OTUs from both libraries into differ-
ent clades was shown in Figs. 3 and 4.
Fig. 2 A comparative bar chart indicating the percentage distribution of clones in different taxa identifi ed in Library I (L-I) and Library
II (L-II)

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174 Indian J Microbiol (June 2009) 49:169–187
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Table 2 Summary of taxonomic assessment of bacterial rDNA sequenced clones from Library I using RDP database
OTUs Clone
name
No. of
clones
Cultured
similarity
score
Accession
no. of
cultured hits
Nearest
phylogenetic
cultured neighbor
Phylum Uncultured
Similarity
Score
Accession no.
of uncultured
hits
Nearest
phylogenetic
uncultured
neighbor
OTU1BE-3731Acidobacteria 0.941AF154083Uncultured
hydrocarbon seep
bacterium BPC102
OTU2BE-0212Gammaproteobacteria0.969DQ396316 Uncultured
organism; ctg_
NISA196
OTU3 BE-1242 Deltaproteobacteria 0.928DQ499326 Uncultured
bacterium; CV106
OTU4 BE-3061 Deltaproteobacteria0.944EF516565 Uncultured
bacterium;
FCPP581
OTU5BE-3383Gemmatimonadetes 0.941 AY913406 Uncultured forest
soil bacterium;
DUNssu200 (-7B)
(OTU#199)
OTU6BE-3831 Acidobacteria0.932 EF125393 Uncultured
bacterium; MSB-
1B7
OTU7 BE-2961 Gammaproteobacteria0.947 AM117932Gamma
proteobacterium
HAL40b
OTU8BE-2941 Acidobacteria 0.945 AF154083 Uncultured
hydrocarbon seep
bacterium BPC102
OTU9BE-04530.823DQ402051
Rhodobacter
azotoformans; S3;
Alphaproteobacteria 0.826AM270417Uncultured
organism; 17H9
OTU10BE-293 10.918 AY987846
Rhodovibrio sp.
2Mb1;
Alphaproteobacteria0.939DQ917822Uncultured
Ochrobactrum sp.;
BME35
OTU11 BE-3361 0.848AJ011330
Phyllobacterium
myrsinacearum;
HM35;
Alphaproteobacteria 0.883DQ395494Uncultured
organism; ctg_
CGOAB08
OTU12BE-32910.92DQ401091
Rhodospirillaceae
bacterium
CL-UU02;
Alphaproteobacteria 0.977 EF157245Uncultured
bacterium; 101-99
OTU13 BE-34110.89 AY741146
Azospirillum
amazonense; 21R;
Alphaproteobacteria 0.922AJ567552 Uncultured alpha
Proteobacterium;
MBMPE38
OTU14 BE-3281 Betaproteobacteria0.977 AB252913Uncultured beta
Proteobacterium;
242
OTU15 BE-3091 Gammaproteobacteria0.95 AY225635Uncultured gamma
Proteobacterium;
AT-s80
OTU16BE-3055 0.912 CP000453
Alkalilimnicola
ehrlichei MLHE-1;
Gammaproteobacteria
OTU17 BE-37410.896 AF304195
Methylobacter
luteus; NCIMB
11914;
Gammaproteobacteria 0.908DQ234131Uncultured
Alcanivorax sp.;
DS047
OTU18BE-38410.883 AY298904
Ectothiorhodosinus
mongolicum; M9;
Gammaproteobacteria 0.905AY328560Uncultured
bacterium;
HOClCi11
OTU19 BE-0802 0.931AF539776
Pseudoalteromonas
sp. R6;
Gammaproteobacteria
OTU20BE-3493 0.897AB246771
Myxobacterium
AT1-01;
Deltaproteobacteria 0.945 DQ811828Uncultured delta
Proteobacterium;
MSB-5C5

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Indian J Microbiol (June 2009) 49:169–187 175
OTUs
name
Clone
name
No. of
clones
Cultured
similarity
score
Accession
no. of
cultured hits
Nearest
phylogenetic
cultured neighbor
Phylum Uncultured
Similarity
Score
Accession no.
of uncultured
hits
Nearest
phylogenetic
uncultured
neighbor
OTU21 BE-0241Firmicutes 0.983AF371787 Uncultured
bacterium; p-2179-
s959-3
OTU22 BE-0042 0.995AJ491303
Caryophanon tenue
(T); type strain:
DSM 14152;
Firmicutes
OTU23BE-0483Actinobacteria 0.946 EF632905Uncultured
bacterium;
Par-s-84
OTU24 BE-3681Actinobacteria0.968DQ395467 Uncultured
organism; ctg_
CGOAA79
OTU25 BE-3081 Actinobacteria0.983AY913337 Uncultured forest
soil bacterium;
DUNssu130 (+7A)
(OTU#190)
OTU26 BE-3313 Actinobacteria 0.895DQ823199 Uncultured
bacterium; ORCA-
17F18
OTU27 BE-0741Actinobacteria 0.945AF317767Unidentifi ed
bacterium
wb1_J07
OTU28BE-0092 TM7 0.924AY930458Uncultured
bacterium; OC28
OTU29BE-3701 unclassifi ed bacteria0.971DQ300602 Uncultured
bacterium; HF130_
C5_P1
OTU30BE-3191 0.937DQ868668
Rhodovulum sp.
SMB1;
Alphaproteobacteria 0.93AM176847 Uncultured
bacterium; SZB36
OTU31BE-365 10.964EF186075
Rhodobacter sp.
DQ12-45T;
Alphaproteobacteria 0.978DQ813946Uncultured
bacterium;
aab57g01
OTU32 BE-09610.964AF136850
Ketogulonicigenium
robustum; X6L;
Alphaproteobacteria 0.959 AF007256 Uncultured alpha
Proteobacterium;
GAI-5
OTU33BE-016 3 0.928AJ401206
Rhodovulum sp.
AT2111;
Alphaproteobacteria0.958EF125428Uncultured
bacterium; MSB-
2C6
OTU34BE-038 20.952AB258386
Kaistobacter terrae;
KCTC12630;
Alphaproteobacteria 0.969AF423277Uncultured soil
bacterium; 565-2
OTU35BE-040 1 0.974AY258089
Mesorhizobium sp.
DG943;
Alphaproteobacteria 0.971 EF125460Uncultured
bacterium; MSB-
2G6
OTU36 BE-0302 0.92 AY654823
Mucus bacterium
86;
Bacteroidetes 0.972 DQ395383Uncultured
organism; ctg_
BRRAA64
OTU37 BE-07010.999DQ480144
Erythrobacter sp.
D3043;
Alphaproteobacteria0.985DQ396252Uncultured
organism; ctg_
NISA320
OTU38BE-00320.974 EF540469
Sphingopyxis sp.
1/4_C7_32;
Alphaproteobacteria0.968AY796041Uncultured
bacterium;
47mm65
OTU39 BE-1095 0.997AJ242582
Ochrobactrum
intermedium;
OiC8-6;
Alphaproteobacteria
0.997 AY851687 Uncultured
Ochrobactrum
sp.; p3
OTU40BE-11046 0.976 DQ660915
Methylophaga sp.
DMS010;
Gammaproteobacteria 0.968DQ490031Uncultured
bacterium; ODP-
46B-02

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123
α-proteobacteria
Out of 14 OTUs (No. 9, 10, 11, 12, 13, 30, 31, 32, 33, 34,
35, 37, 38 and 39) (27.45%) belonging to α-proteobacteria
of L-I, except 1 OTU (OTU9), all are clustered with α-
proteobacteria clade supported by 100 bootstrap value.
Three OTUs (No. 10, 12 and 13) of α-proteobacteria
clustered with Rhodospirillaceae bacterium CL-UU02
an isolate from urea-enriched seawater (Cho et al. un-
published, DQ401091). OTU35 of L-I had high 97.4%
sequence identity to Mesorhizobium sp. DG943 an isolate
from the dinofl agellate Gymnodinium catenatum known for
paralytic shellfi sh poisoning [25]. OTU37 of L-I had very
high 99.9% sequence identity to Erythrobacter sp. D3043
an isolate from Qing Dao Coast, an anoxygenic phototroph
having large amount of carotenoides, it plays important role
in cycling of both organic and inorganic carbon in the ocean
(Li et al. Unpublished, DQ480144 and [26]). OTU39 of L-
I with 5 clones had very high 99.7% sequence identity to
Ochrobactrum intermedium, an emerging human pathogen
among the liver abscess, post-liver transplantation and in
the bladder cancer patients, causing presumptive bacte-
remia [27]. OTU32 of L-I had 96.4% sequence identity
to Ketogulonogenium robustum which had the ability to
produce vitamin C from substrates like L-sorbosone [28].
Phylogenetic clustering of above OTUs with there clos-
est homologs organism was supported by 100 bootstrap
value. OTU31 clustered with Rhodovibrio sp. 2Mb1, an
isolate from maras salterns, a hypersaline environment in
the peruvian andes [29]. OTU9 of L-I with three clones had
only 82.3% sequence identity to Rhodobacter azotoformans
strain S3, denitrifying phototrophic bacteria holding ability
to reduce selenite to red elemental selenium [30]. This OTU
had very low sequence similarity with α-proteobacteria.
Phylogenetically this OTU clustered with Myxobacterium
(AB246771), a δ-proteobacteria with 100 bootstrap confi -
dence value. α-proteobacteria group of L-II had 6 OTUs
(No. 5, 6, 7, 8, 9 and 10) (11.76%) with 12 clones. All these
OTUs Clone
name
No. of
clones
Cultured
Similarity
Score
Accession
no. of
cultured hits
Nearest
phylogenetic
cultured neighbor
PhylumUncultured
Similarity
Score
Accession no.
of uncultured
hits
Nearest
phylogenetic
uncultured
neighbor
OTU41BE-343 100.964DQ660915
Methylophaga sp.
DMS010;
Gammaproteobacteria0.959 DQ513013Uncultured
bacterium; FS140-
15B-02
OTU42BE-0581 0.977 AB167031
Marinobacter sp.
NT N115;
Gammaproteobacteria0.984AF513454 Alteromonadaceae
bacterium LA50
OTU43BE-3721 0.999 X74677
Aeromonas
hydrophila subsp.
hydrophila (T);
ATCC 7966T;
Gammaproteobacteria
OTU44BE-1411 0.993AB021194
Bacillus niacini (T);
IFO15566;
Firmicutes 0.995 AY642567Uncultured low
G+C Gram-positive
bacterium; LV60-
10
OTU45BE-010 2 Acidobacteria 0.967 DQ378243Uncultured soil
bacterium; M23_
Pitesti
OTU46 BE-3712Acidobacteria0.951 DQ395012Uncultured
Acidobacteriaceae
bacterium; VHS-
B4-48
OTU47 BE-3561Verrucomicrobia 0.929EF516510Uncultured
bacterium;
FCPN572
OTU48 BE-0883 0.983X87339
Methylophaga
thalassica (T);
ATCC 33146;
Gammaproteobacteria0.977DQ513013 Uncultured
bacterium; FS140-
15B-02
OTU49 BE-0711 0.956 AB053124
Alcanivorax sp.
Mho1;
Gammaproteobacteria0.96 DQ234131Uncultured
Alcanivorax sp.;
DS047
OTU50BE-082 120.99 AB055205
Alcanivorax sp.
K3-3; K3-3 (MBIC
4323);
Gammaproteobacteria0.978 DQ396108Uncultured
organism; ctg_
NISA076
OTU51BE-33410.944 DQ084461
Pseudomonas sp.
FLM05-3;
Gammaproteobacteria0.944 AY569287 Uncultured
Pseudomonas sp.;
YJQ-10

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Indian J Microbiol (June 2009) 49:169–187 177
Table 3 Summary of taxonomic assessment of bacterial rDNA sequenced clones from Library II using RDP database
OTUsClone
name
No. of
clones
Cultured
similarity
score
Accession no.
of cultured
hits
Nearest
phylogenetic
cultured neighbor
PhylumUncultured
Similarity
Score
Accession
no. of
uncultured
hits
Nearest phylogenetic
uncultured neighbor
OTU1 NE-1001 0.96AF170424 Sulfur-oxidizing
bacterium NDII1.1
Gammaproteobacteria 0.961 DQ811847 Uncultured gamma
Proteobacterium;
MSB-5C2
OTU2 NE-2572 0.984Z67753
Odontella sinensis
Cyanobacteria 0.991DQ521522 Uncultured bacterium;
ANTLV2_F08
OTU3 NE-279 1 Chlorofl exi 0.95DQ154828 Uncultured bacterium;
GN01-8.065
OTU4 NE-912 0.884 DQ660913
Methylophaga sp.
DMS004
Gammaproteobacteria 0.878DQ490031Uncultured bacterium;
ODP-46B-02
OTU5 NE-166 10.948 AY654775
Mucus bacterium 32
Alphaproteobacteria 0.955 DQ153134 Uncultured alpha
Proteobacterium;
06-03-45
OTU6NE-115 0.954AF513400
Rhodobacter sp.
Alphaproteobacteria0.968 AY258094Bacterium DG981
OTU7NE-211 20.958AM696304
Rhodovulum
marinum
Alphaproteobacteria 0.948 AY345479Bacterium K2-91B
OTU8 NE-3662 0.934 DQ868668
Rhodovulum sp.
Alphaproteobacteria 0.958 AJ567557Uncultured alpha
Proteobacterium;
MBMPE43
OTU9NE-1501 0.962 DQ868668
Rhodovulum sp.
Alphaproteobacteria0.972 DQ811853Uncultured alpha
Proteobacterium;
MSB-3E4
OTU10 NE-3731 0.975AM712634
Anderseniella
baltica; type strain:
BA141
Alphaproteobacteria 0.998 EF061946 Uncultured alpha
Proteobacterium;
XME30
OTU11NE-534 0.97 CP000284
Methylobacillus
fl agellatus KT
Betaproteobacteria0.96AJ582036Uncultured beta
Proteobacterium;
JG36-GS-101
OTU12 NE-3022 0.941AB089481
Derxia gummosa
Betaproteobacteria 0.982AB286331 Uncultured bacterium;
0101
OTU13 NE-303 3Gammaproteobacteria 0.985DQ351809Uncultured gamma
Proteobacterium;
Belgica2005/10-ZG-17
OTU14NE-330 3Gammaproteobacteria 0.992EF125435Uncultured bacterium;
MSB-2D6
OTU15NE-3492 0.937EF117913
Thiohalomonas
denitrifi cans
Gammaproteobacteria 0.945AY225635 Uncultured gamma
Proteobacterium;
AT-s80
OTU16NE-1391Gammaproteobacteria 0.979 EF208680Uncultured bacterium;
CI5cm.B02
OTU17
NE-10510.983 AB021367
Marinobacterium
stanieri (T); ATCC
27130T
Gammaproteobacteria 0.959EF190069Uncultured
Marinobacterium sp.;
GSX2
OTU18 NE-2672 Gammaproteobacteria0.989 EF208690 Uncultured bacterium;
CI5cm.F08
OTU19 NE-132 0.946AB053125
Alcanivorax sp.
Gammaproteobacteria 0.936AJ567576 Uncultured
Marinobacter sp.;
MBAE14
OTU20 NE-2110.894 AJ237601
Desulfobacterium
anilini (T); DSM
4660
Deltaproteobacteria 0.975 DQ463694Uncultured delta
Proteobacterium;
TK-SH10
OTU21NE-292 4Deltaproteobacteria0.984 AJ535245 Uncultured delta
Proteobacterium
OTU22NE-114 10.916EF422413
Desulfurivibrio
alkaliphilus
Deltaproteobacteria 0.944 DQ831546Uncultured delta
Proteobacterium;
CBII30
OTU23 NE-4310.871 AY187308
Pelobacter
masseliensis
Deltaproteobacteria 0.929EF999371 Uncultured bacterium;
MidBa15

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178 Indian J Microbiol (June 2009) 49:169–187
123
OTUsClone
name
No. of
clones
Cultured
Similarity
Score
Accession no.
of cultured
hits
Nearest
phylogenetic
cultured neighbor
Phylum Uncultured
Similarity
Score
Accession
no. of
uncultured
hits
Nearest phylogenetic
uncultured neighbor
OTU24 NE-240 10.923X94911
Syntrophobacter sp.
Deltaproteobacteria 0.964DQ811826 Uncultured delta
Proteobacterium;
MSB-5bx5
OTU25 NE-3783 0.956 AJ620511
Olavius
crassitunicatus
delta-proteobacterial
endosymbiont;
d3-P12-1
Deltaproteobacteria 0.991 DQ811824Uncultured delta
Proteobacterium
MSB-5B3
OTU26NE-3721 0.913 AY493563 Sulfate-reducing
bacterium
Deltaproteobacteria 0.923U81720Uncultured
Eubacterium;
vadinHA60
OTU27NE-291 10.92 CP000482
Pelobacter
propionicus DSM
2379
Deltaproteobacteria 0.92 AF529129 Uncultured delta
Proteobacterium;
FTLpost101
OTU28NE-191 0.973 AJ271656
Pelobacter sp. A3b3
Deltaproteobacteria 0.974AJ240980Uncultured delta
Proteobacterium
Sva1034
OTU29 NE-364 1Actinobacteria 0.979 AM259898 Uncultured
Actinobacterium;
TAA-10-01
OTU30NE-2511 Actinobacteria 0.978 DQ070822
Uncultured Gram-
positive bacterium;
JdFBGBact_23
OTU31NE-821Acidobacteria0.979DQ395006 Uncultured
Acidobacteria
bacterium; VHS-B4-69
OTU32 NE-371 0.905AJ784892
Haliscomenobacter
hydrossis; DSM
1100
Bacteroidetes 0.916 EF508145 Uncultured
Sphingobacterium sp.;
MS190-1F
OTU33 NE-3761Bacteroidetes 0.939 AJ567581 Uncultured
Bacteroidetes
bacterium; MBAE20
OTU34 NE-70 2 0.907AB331889
Verrucomicrobia
bacterium YM27-
120
Verrucomicrobia 0.904AY345492Unidentifi ed
bacterium; W4-B59
OTU35NE-5 10.94 AF165908
Escarpia spicata
endosymbiont
‘Alvin #2839
Gammaproteobacteria 0.948AB278144 Uncultured bacterium;
Mafs-EB04
OTU36NE-2523 0.923 AF328856
Olavius algarvensis
sulfur-oxidizing
endosymbiont;
126I-9
Gammaproteobacteria 0.938DQ811837 Uncultured gamma
Proteobacterium;
MSB-3A4
OTU37 NE-3411 Gammaproteobacteria 0.944DQ351759 Uncultured gamma
Proteobacterium;
Belgica2005/10-
130-14
OTU38
NE-273310.972 X95460
Methylophaga
thalassica; SM5690
Gammaproteobacteria 0.971DQ490031Uncultured bacterium;
ODP-46B-02
OTU39 NE-2849 0.966 DQ660929
Methylophaga sp.
DMS044
Gammaproteobacteria 0.965 DQ490031Uncultured bacterium;
ODP-46B-02
OTU40 NE-21914 0.962 DQ660930
Methylophaga sp.
DMS048
Gammaproteobacteria 0.962DQ490031Uncultured bacterium;
ODP-46B-02
OTU41NE-1404 0.988DQ458821
Marinobacter sp.
HS225
Gammaproteobacteria
OTU42NE-2932Acidobacteria 0.938 EF125465Uncultured bacterium;
MSB-2Y4
OTU43 NE-921 Acidobacteria 0.952AJ241003Uncultured holophaga/
Acidobacterium
Sva0725

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Indian J Microbiol (June 2009) 49:169–187 179
OTUs form a separate cluster of α-proteobacteria sup-
ported by 100 bootstrap value and grouped with there near-
est cultured and uncultured homologs from the database.
OTU5 had 94.8% sequence identity to Mucus bacterium
32 isolated from the mucus of Oculina patagonica (Koren
et al. Unpublished, AY654823). OTU6 with 5 clones had
95.4% similarity to Rhodobacter sp. 1-5, an isolate from
Arctic sea ice, Spitzbergen. OTU7 with 2 clones had 95.8%
sequence identity to Rhodovulum marinum strain JA242,
isolated from different habitats of India [31], it contain
bacteriochlorophyll-a and carotenoides in vesicular intracy-
toplasmic membranes. OTU8 and OTU9 with 2 and 1 clone
had 93.4 and 96.2% sequence identity respectively to Rhod-
ovulum sp. SMB1 (Choong et al. unpublished, DQ868668).
OTU10 had high 97.5% sequence identity to Anderseniella
baltica type strain BA141T, isolated from sediment in the
central Baltic Sea characterized by the presence of carot-
enoides and absence of bacteriochlorophyll a [32].
β-proteobacteria
β-proteobacteria had constituted only 1 OTU from L-I,
which had not shown any similarity to cultured sequences.
Phylogenetically it forms sister clade with γ-proteobacteria
with 97 bootstrap value where as L-II is represented with 2
OTUs (No. 11, 12). OTU11 of L-II with 4 clones had high
sequence identity of 97% to Methylobacillus fl agellatus KT
(Copeland et al. unpublished, CP000284). OTU12 of L-II
with 2 clones had 94.1% sequence identity to Derxia gum-
mosa strain: IAM14990. Derxia is a nitrogen-fi xing genus
identifi ed with a bacterium isolated from West Bengal soil
of India [33].
δ-proteobacteria
δ-proteobacteria with 3 OTUs (No. 3, 4 and 20) (5.88%)
containing 6 clones had constituted 4% part of L-I, OTU20
with 3 clones had 89.7% sequence identity to Myxobac-
terium AT1-01 an isolate from hot springs in Japan [34].
δ-proteobacteria group of L-II had 12 OTUs (No. 20, 21,
22, 23, 24, 25, 26, 27, 28, 49, 50 and 51) (23.52%) with 18
clones. OTU20 and OTU50 had 89.4 and 95.5% sequence
identity to Desulfobacterium anilini strain DSM 4660, sul-
fate-reducing bacteria capable of aniline and dihydroxyben-
zenes degradation isolated from marine sediment [35, 36].
OTU22 had 91.6% sequence identity to Desulfurivibrio
alkaliphilus strain AHT2, halophilic bacteria of reductive
sulfur cycle from Soda Lake (Sorokin et al. unpublished,
EF422413). OTU24 had 92.3% sequence identity to
Syntrophobacter sp. which is a syntrophic propionate-
oxidizing, sulfate-reducing bacterium from a fl uidized bed
reactor [37]. OTU25 with 3 clones had 95.6% identity to
Olavius crassitunicatus clone d3-P12-1, an endosymbiont
of a gutless worm (Oligochaeta) from the Peru margin, ca-
pable of sulfi de oxidation [38]. OTU26 had 91.3% sequence
identity to sulfate-reducing bacterium PF2802 capable of
OTUsClone
name
No. of
clones
Cultured
Similarity
Score
Accession no.
of cultured
hits
Nearest
phylogenetic
cultured neighbor
PhylumUncultured
Similarity
Score
Accession
no. of
uncultured
Hits
Nearest phylogenetic
uncultured neighbor
OTU44 NE-1301 Acidobacteria 0.952 EF125465Uncultured bacterium;
MSB-2Y4
OTU45NE-1071Acidobacteria 0.965 DQ351815Uncultured
Acidobacteriales
bacterium;
Belgica2005/10-ZG-24
OTU46 NE-374 1Acidobacteria 0.968AB294930 Uncultured
Acidobacteria
bacterium;
pItb-vmat-12
OTU47 NE-752Verrucomicrobia 0.957AY114336 Uncultured
Verrucomicrobia
bacterium; LD1-PB9
OTU48NE-226280.971 X87339
Methylophaga
thalassica (T); TCC
33146;
Gammaproteobacteria 0.965 DQ490031Uncultured bacterium;
ODP-46B-02
OTU49 NE-441Deltaproteobacteria 0.938 AY375087Uncultured bacterium;
C10;
OTU50NE-631 0.955 AJ237601
Desulfobacterium
anilini (T); DSM
4660
Deltaproteobacteria 0.995 DQ811831 Uncultured delta
Proteobacterium;
MSB-5D12;
OTU51 NE-2662 Deltaproteobacteria 0.986DQ811801 Uncultured delta
Proteobacterium;
MSB-3E9;

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180 Indian J Microbiol (June 2009) 49:169–187
123
Fig. 3 Unrooted phylogenetic dendogram for Library I (L-I) dataset was drawn using bayesian inference method with program MrBayes
3.0. The analysis for 16S rRNA gene consisted of 3,000,000 generations. An appropriate model of sequence evolution for data set was
chosen via Akaike Information Criterion (AIC) using program MrModeltest 2.2 and the model selected was (GTR+I+G). Trees were
sampled for every 100 generations. First 3000 trees (10%) were discarded as burnin. Bayesian posterior probabilities were calculated using
50% majority rule consensus and three independent runs were performed. Text in parentheses indicate the representative clone name.

Page 13

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Indian J Microbiol (June 2009) 49:169–187 181
Fig. 4 Unrooted phylogenetic dendogram for Library II (L-II) dataset was drawn using bayesian inference method with program
MrBayes 3.0. The analysis for 16S rRNA gene consisted of 3,000,000 generations. An appropriate model of sequence evolution for data set
was chosen via Akaike Information Criterion (AIC) using program MrModeltest 2.2 and the model selected was (GTR+I+G). Trees were
sampled for every 100 generations. First 3000 trees (10%) were discarded as burnin. Bayesian posterior probabilities were calculated using
50% majority rule consensus and three independent runs were performed. Text in parentheses indicate the representative clone name.

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182 Indian J Microbiol (June 2009) 49:169–187
123
degrading n-alkene (Cravo et al. unpublished, AY493563).
OTU27 and OTU28 had 92% and 97.3% sequence identity
to Pelobacter propionicus DSM 2379 and Pelobacter sp.
clone A3b3 respectively, both found in marine sediments
and capable of sulphate reduction (unpublished, CP000482
and [39]). Phylogenetically, all these OTUs clustered with
the δ-proteobacteria clade.
γ-proteobacteria
Majority of OTUs (29.4%) belonging to γ-proteobacteria in
L-I had shown affi liation to cultured isolates but with very
less similarity. Phylogenetically all OTUs clustered with
γ-proteobacteria clade showing 100 bootstrap confi dence
value. OTU16 with 5 clones had 91.2% sequence identity to
Alkalilimnicola ehrlichei MLHE-1, an anaerobic, faculta-
tively autotrophic arsenite oxidizing bacterium that respires
nitrate or nitrite (Copeland et al. unpublished, CP000453
and [40]). OTU17 of L-I had 89.6% sequence identity to
Methylobacter luteus NCIMB, a methanotrophic bacteria
with an ability to consume nitric oxide and produce small
amounts of nitrous oxide [41]. OTU18 of L-I had 88.3%
sequence identity to Ectothiorhodosinus mongolicum strain
M9, a purple sulfur bacterium isolated from a Mongolian
soda lake having photosynthetic pigments such as bac-
teriochlorophyll a and carotenoids of the spirilloxanthin
series (Gorlenko et al. unpublished, AY298904). OTU40 of
L-I with 46 clones and OTU41 of L-I with 10 clones were
among the most abundant group, they had high 97.6 and
96.4% sequence identity respectively to Methylophaga sp.
DMS010, an isolate from marine dimethylsulfi de-degrad-
ing enrichment. It carries out oxidation of DMS by a route
different from those described for Thiobacillus species [42].
OTU43 of L-I had very high 99.9% sequence identity to
Aeromonas hydrophila (ATCC 7966T) which cause infec-
tions in invertebrate and vertebrate such as frogs, birds
and domestic animals, also an emerging human pathogen
irrespective of the host’s immune system [43]. OTU48 of
L-I had high 98.3% sequence identity to Methylophaga
thalassica (T); ATCC 33146 it also had an ability to degrade
dimethysulfi de. OTU49 and OTU50 of L-I with clones 1
and 12 had high sequence identity of 95.6 to 99% to
Alcanivorax sp. Mho1 and Alcanivorax sp. K3-3 respec-
tively, these are alkane-degrading marine bacterium pre-
dominated in petroleum or hydrocarbon contaminated sea-
water or sediments (Kishira et al. unpublished, AB053124
and [44]). Phylogentic clustering of these OTUs with
respective cultured homologs is supported by higher node
value. γ-proteobacteria was also the most abundant taxon
in Library-II. Most of the OTUs (33.34%) had shown affi li-
ation to cultured representatives. OTU1 had high sequence
identity of 96% to sulfur-oxidizing bacterium NDII1.1 an
isolate from a shallow water hydrothermal vent (Sievert
et al. unpublished, AF17042). 5 OTUs (No. 4, 38, 39, 40
and 48) of L-II with clones 2, 31, 9, 14 and 28 had high
88.4 to 98.8% sequence identity to Methylophaga sp. and
Methylophaga thalassica clustered with this organism with
100 bootstrap value. Methylophaga sp. was the most abun-
dant homolog in both the libraries. OTU15 with 2 clones
had 93.7% sequence identity to Thiohalomonas denitrifi -
cans strain HLD 14, a thiodenitrifying halophilic bacteria
isolated from sediments of a solar saltern (Tourova et al.
unpublished, EF117913). OTU19 of L-II with 2 clones had
94.6% sequence identity to Alcanivorax sp. I4 (Kishira et
al. unpublished, AB053125), L-I had a rich representation
of these sequences with 13 clones. OTU35 of L-II had se-
quence identity of 94% to Escarpia spicata endosymbiont
Alvin #2839 an isolate from vestimentiferan tubeworms
[45]. OTU36 of L-II with 3 clones had 92.3% identity to
Olavius algarvensis, a sulfur-oxidizing endosymbiont from
an oligochaete worm [46]. OTU41 of L-II with 4 clones
had high 98.8% identity to Marinobacter sp. HS225 strain
HS225, a halophile isolated from the Zhoushan Archipelago
of China (Xu et al. unpublished, DQ458821).
Acidobacteria
5 OTUs (No. 1, 6, 8, 45 and 46) (9.8%) from L-I showed
close relationships to each other and formed one large clus-
ter having three sister clade with 99 bootstrap confi dence
value. The closest species from database to the above group
are uncultivated bacteria from oil polluted soil of Roma-
nia, stromatolites of Hamelin pool in Shark Bay, western
Australia, near shore and outer shelf reefs of Australia,
Fushan forest soils of Taiwan and hydrocarbon seep sedi-
ments. Similarly 6 OTUs (No. 31, 42, 43, 44, 45 and 46)
(11.76%) of L-II formed a cluster and were closely related
to uncultured Holophaga/Acidobacteria from Great Bar-
rier Reef calcareous sediments of Australia, mangrove soil,
permeable shelf sediments, organically-enriched fi sh farm
sediments and hydrothermal sediments.
Actinobacteria
5 OTUs (No. 23, 24, 25, 26 and 27) (9.8%) were clustered
to Actinobacteria from L-I. They formed a separate cluster
with 98 bootstrap value and showed homology to a number
of uncultured Actinobacteria from a variety of places like
perennial ice cover of Lake Vida, Antarctica, Nullabar caves,
Oregon caves, Hypersaline Gulf of Mexico sediments and
deep sea. Only 2 OTUs (No. 29, 30) (3.92%) from L-II were
included in Actinobacteria and both were closely related to
uncultured Actinobacteria with 100% node value.

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Indian J Microbiol (June 2009) 49:169–187 183
Firmicutes
Firmicutes forms a separate cluster, constituted 3 OTUs
(5.88%) with 100 node value from L-I. OTU22 containing
2 clones, had very high 99.5% sequence identity to Caryo-
phanon tenue type strain DSM 14152T (Fritze, D. unpub-
lished, AJ491303). OTU44 of L-I had high 99.3% sequence
identity to Bacillus niacini which produce an ofl oxacin
ester-enantioselective estrase [47].
Bacteroidetes
Bacteroidetes had almost equal representation among both
the libraries. Bacteroidetes in L-I is presented by OTU36
with 2 clones grouped with Mucus bacterium 86 having
92% sequence identity, it is isolated from the mucus of
Oculina patagonica a scleractinian coral (Koren et al. Unpub-
lished, AY654823). OTU32 and OTU33 of L-II had shown
clustering with Haliscomenobacter hydrossis strain DSM
1100, isolated from the pelagic zones of a broad spectrum
of freshwater habitats. H. hydrossis has one of the smallest
widths of any of the fi lamentous bacteria, 0.5 μm, and fi la-
ment usually extending to a length of 20–100 μm [6].
Verrucomicrobia
Verrucomicrobia sequence from L-I does not show similar-
ity to any cultured bacteria, and it form a sister clade with
the Firmicutes with 100 bootstrap value. Verrucomicrobia
is represented in L-II with 2 OTUs and 4 clones, constitut-
ing 2.46% of total clones. OTU34 and OTU47 of L-II had
90.7% similarity to cultured Verrucomicrobia bacterium
YM27-120 (Matsuo et al. unpublished, AB 331889). These
OTUs form a sister clade with Cyanobacteria.
Other minor phylogenetic groups
These groups had a minor contribution to the bacterial
diversity studied. It includes Chlorofl exi, Cyanobacteria,
Gemmatimonadetes, Candidate division TM7 and unclas-
sifi ed bacteria. OTUs belonging to Gemmatimonadetes
and unclassifi ed bacteria from L-I form a cluster with 100
bootstrap confi dence value, suggesting that this unclassifi ed
OTU may belongs to Gemmatimonadetes taxon. Phyloge-
netically Candidate division TM7 clustere with Verruco-
microbia supported by lower bootstrap value. Candidate
division TM7 are named after sequences obtained in an
environmental study of a peat bog [21], later on partial-
length-sequence representatives of this Candidate divi-
sion were subsequently identifi ed from activated sludges
and soil. We found OTU3 clustering with Chlorofl exi and
OTU2 with Cyanobacteria in L-II. OTU2 showed cluster-
ing to Odentella sinensis with 93 bootstrap value, and were
derived from the chloroplast of an alga [48] and were most
closely related to the Cyanobacteria.
Statistical analyses: species richness
Statistical analyses of biodiversity provide interesting in-
sights (Table 4). 29 singleton OTUs (OTUs having single
clone) with Good’s coverage 80.41% in L-I and 27 single-
tons OTUs with Good’s coverage 83.34% in L-II suggested
that the coverage was fi ne but it also indicated that any new
clone had 19.59% and 16.66% of chance to fall in an un-
known species from respective libraries. The higher value
of Shannon index (3.1679) in L-II suggest higher diversity,
richness and even distribution of abundance, indicating that
species are more diverse and evenly distributed in L-II as
compared to L-I. The value of Simpson index for library I
(0.1100) suggest that probability of fi nding a new phylo-
type is more in L-I than L-II (0.0784). Rarefaction analysis
shows a curvilinear plot but it did not plateau with the cur-
rent coverage. The curve (Fig. 5) indicates that the diversity
was sampled with good level of confi dence and majority
of OTUs in the sample were detected but still there is a
need of more comprehensive sampling to cover rest of the
undiscovered diversity. Signifi cant P = 0.05 decrease in the
rate of OTU detection with increasing number of clones ex-
amined was observed in rarefaction curves of both libraries.
Chao values suggest undiscovered OTUs/phylotypes in the
library which could be still explored by more robust mehod.
∫-LIBSHUFF with 10,000 randomization found P value
(0.001) for comparison, which provided strong evidence
that none of the library is a subset of the other and two
libraries are considered signifi cantly different communities.
Discussion
Study of marine microbial biodiversity is of vital impor-
tance to the understanding of the different processes of the
Table 4 Statistical Indices calculated for two libraries
Library ILibrary II
No. of sequences 148162
No. of OTUs* 51 51
Singletons 2927
Shannon Index 3.0918 + 0.2479
3.1679 + 0.2123
Simpson Index0.110039 0.07836
Chaol87.909178
Good’s coverage80.41% 83.34%
* 97% similarity clusters have been considered as operational
taxonomic units (OTUs)