Cultivation and Characterization of Microorganisms in Antarctic Lakes
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Microorganisms were isolated from Antarctica lakes and
identified based on 16S rDNA sequence analysis. The lake samples
were collected from Antarctic lakes in Skavrvsnes near Syowa
Station area. When cultivation of lake water was performed at 4
ºC and 20 ºC in several selection media, most of microorganisms
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Cultivation and Characterization of Microorganisms
in Antarctic Lakes
Hongyan Zhang
1
, Shoko Hosoi-Tanabe
1
*, Shinichi Nagata
1
, Syuhei Ban
2
, and Satoshi Imura
3
1
Research Center for Inland Seas, Kobe University, 5-1-1 Fukaeminami, Higashinada, Kobe 658-0022, Japan
2
School of Environmental Science, University of Shiga Prefecture, Hassaka-cho, Hikone, Shiga 522-8533, Japan
3
National Institute of Polar Research 9-10, Kaga 1-chome, Itabashi-ku, Tokyo 173-8515, Japan
*Corresponding author; E-mail: syonatsu@maritime.kobe-u.ac.jp
Abstract
Microorganisms were isolated from Antarctica lakes and
identified based on 16S rDNA sequence analysis. The lake samples
were collected from Antarctic lakes in Skavrvsnes near Syowa
Station area. When cultivation of lake water was performed at 4
ºC and 20 ºC in several selection media, most of microorganisms
could not grow at 20 ºC, but they could efficiently grow at 4 ºC,
indicating that low incubation temperature was more suitable for
microorganisms in Antarctica lake. 16S rDNA of each isolate was
sequenced. Homology search on GenBank showed that some clones
were closely related to class of Flavobacteria, Bacilli, Actinobacteria,
Alpha-proteobacteria, and Gamma-proteobacteria. A strain which
grew at 20 ºC was identical to previously characterized bacterium
(Gillisia limnaea) with 93% of 16S rDNA similarity, which
suggested that this strain might be representative of novel genera.
I. INTRODUCTION
Antarctic zone pristine biotopes are subject to long periods
of ice and snow-cover, low temperatures, and low levels of
photosynthetically active radiation. As several research
techniques including the skill of crossing an Antarctic sea and
land, sampling system, and analysis method are advancing in
recent year, psychrotrophs/psychrophiles have been understood
in these permanent cold environments. Especially, the development
of molecular biological techniquehave contributedtothe understanding
a certain knowledge of these organisms’ characteristics, and a
lot of studies about biological diversity using this technique
without cultivation have been accumulated in last decades,
overcoming the inability to cultivate the majority of
microorganisms from environmental samples [1,2]. However,
this approach also has limitation because only sequence data
is clarified.
Therefore, cultivating and characterizing isolates are also
thought to be particularly important for providing a more
comprehensive view of diversity because these analyses make
the characteristics of psychrotrophs/ psychrophilesmicroorganisms
clear, and we might get several interesting information such as
the low temperature adaptability of organisms. Moreover, some
psychrotrophs/ psychrophilesmicroorganisms’application toindustry
might develop commercial product working at low temperature.
Unfortunately, the recovery of culturable organisms from Antarctica
is very difficult, and the development of new methods to resuscitation
and culturability of Antarctic microorganisms is very important.
Inorder tounderstandthediversity, survival,and activityofmicroorganisms
in Antarctic zone, we cultivated and characterized bacterial isolates
from Antarctic lakes.
II. MATERIALS AND METHODS
Sampling site
Water samples were collected from Antarctic lakes in
Skavrvsnes near Syowa Station area (A-6 Ike, depth 0 m,
sample date 19th January 2005; A-7 Ike, depth 0 m, sample
date 29th January 2005; B-1 Ike, depth 0 m, sample date 21th
January 2005; B-3 Ike, depth 0 m, sample date 21th January
2005; Hunazoko Ike, depth 4 m, 22th January 2005; Tokkuri
Ike, depth 4 m, sample date 22th January 2005; Suribati Ike,
depth 10 m, sample date 24th January 2005 in Fig.1).
Media
LB Agar medium (g/liter): Bacto tryptone 10; Bacto-yeast
extract 5; Agar 15 with different NaCl concentration (0 M, 1
M, 2 M, 3 M, 4 M) [3]. Beef extract Agar medium (g/liter):
Beef extract 10; Peptone 10; Agar 15 with different NaCl
concentration (0 M, 1 M, 2 M, 3 M, 4 M) [3]. R2A Agar
medium (g/liter): Yeast extract 0.5; Protease Peptone no. 3 0.5;
Casamino Acids 0.5; Glucose 0.5; Soluble starch 0.5; Sodium
pyruvate 0.3; K
2
HPO
4
0.3; MgSO
4
·7H
2
O 0.05; Agar 15.0 [3].
YM Agar medium (g/liter): Yeast extract 3; Malt extract 3;
Peptone 5; Glucose 10; Agar 15 with different NaCl
concentration (0 M, 1 M, 2 M, 3 M, 4 M) [3]. PYMV Agar
medium (g/liter): Mineral salt sol. ("Hutner / Cohen-Bazire")
20 ml; Peptone 0.25; Yeast extract 0.25; Agar 15; Glucose sol.
(2.5%, sterile-filtered) 10 ml; Vitamin sol. (double conc.) 5 ml;
Adjust pH to 7.5 (the medium is only weakly buffered; one
needs approx. 10 drops/liter medium of 6 N KOH) [3].
Culture condition
Antarctic samples collected in 2005 from 7 lakes were
directly spread on several selection agar-media (LB Agar
medium, Beef extract Agar medium, and YM Agar medium),
developed for bacterial isolation, and incubated at 20 ºC for
15 days. Some samples were concentrated with 0.2 µm
Nuclepore Track-Etch Membrane filtration products (Whatman Inc.,
Kent, UK), washed with PBS Buffer (pH 7.4), spread on
several selection agar-media (R2A Agar medium, PYMV
Agar medium) and incubated at 4 ºC for 2 months. The grown
bacterial colonies on each media were re-streaked several times,
incubated by two selection media, R2A and PYMV specific for
eutrophic bacterial isolation, at 20 °C for up to 20 days, and
developed as isolates [3-5].
Preparation of DNA and PCR
Colonies of each isolate were suspended in 20 µl of TE
buffer (pH 8.0), boiled for 10 minutes and placed on ice for 5
minutes. The solutions were used as DNA template solutions
for amplification of 16S rDNA.
Partial 16S rDNA sequences (about 500 bp) were amplified
by PCR with the common primers EUB 27F 5'-AGAGT
TTGATCMTGGCTCAG-3' [6] and EUB 533R: 5'-TTACC
GCGGCKGCTGRCAC-3' [6]. PCR was performed in 20 µl
volumes containing 1.25 u of Go Taq polymerase (Promega,
Madison, USA), 1×Green Go Taq Reaction Buffer, 0.2 mM
dNTPs, 0.2 µM of each primers set, and 1 µl of template DNA
under the conditions: heating at 94 °C for 5 minutes, followed
by 30 cycles of denaturation at 94 °C for 30 seconds, annealing
at 50 °C for 30 seconds, and elongation at 72 °C for 1 minute.
After cycling, extension was carried out at 72 ºC for 10 minutes
[4].
Cloning
The PCR products were purified with the Wizad SV Gel
and PCR Clean-up System Kit (Promega, Madison, USA) for
use as sequencing templates. The 16S rDNA amplicons were
sequenced, using the ABI Prism Big Dye Terminator Cycle
Sequencing Ready Reaction Kit on an ABI 3100 DNA sequencer
(Applied Biosystems, Foster City, CA, USA).
The 16S rDNA sequences were examined for similarity
using FASTA [7] and BLAST [8, 9] at the DNA Data Bank of
Japan (DDBJ, http://www.ddbj.nig.ac.jp) and National Center
forBiotechnologyInformation(NCBI,http://www.ncbi.nlm.nih.gov/bl2seq).
III. RESULT AND DISCUSSION
CultivationofAntarcticlakesamplesandisolationofmicroorganisms
After first incubation, we could recover 22 bacterial strains
(strain No. 15, 20, 22-33, 35-37, and 41-45) on the basis of the
difference of colony size, shine and color. While twenty bacteria
strains (strain No. 22-33, 35-37, and 41-45) were isolated from
incubation at 4 °C onlytwo bacteria strains (strain No. 15, 20) were
isolated from incubation at 20 °C. Different colorful colonies
(orange, white, yellow, buff, and sandy beige) were appeared
on incubation agar-media, and some strains (strain No. 15, 41)
had a slimy and gummy appearance. As one of these reasons
that there were onlytwo strains isolated from incubation at 20 °C
A
B
Fig.1 A.The location of Skavrvsnes; B. Lakes on Skavrvsnes.1. Hunazoko Ike, 4.Tokkuri Ike, 9.B1 Ike, 11. B3 Ike, 20.A6 Ike, 21. A7 Ike, 30. Suribati Ike.
TABLE I
HOMOLOGY ANALYSISOFISOLATESFROMANTARCTICAA-6 IKE,A-7 IKE,B-1 IKE,B-3 IKE,SURIBAT I IKE,TOKKURI IKE,AND HUNAZOKO IKE
16S rDNA
Strain
No.
Lake
Close representative
Organisms/ group
Sequence
accession number
Similarity
(%)
E-Value
Incubation
temperature
(°C)
Colony
description
15
Hunazoko Ike
Gillisia limnaea (R-8282)
AJ440991
93
0.0
20
Orange, slimy
20
B-1 Ike
Gracilibacillus halotolerans (NN)
AF036922
99
0.0
20
White
22
A-6 Ike
Psychrobacter fozii (S2-83)
AY771717
99
0.0
4
White
23
Tokkuri Ike
Psychrobacter maritimus (Pi2-20T)
AJ609272
99
0.0
4
White
24
B-3 Ike
Psychrobacter alimentarius (S3-15)
AY771725
99
0.0
4
White
25
Tokkuri Ike
Flavobacterium frigidarium (S3-9)
AY771722
99
0.0
4
Yellow
26
Tokkuri Ike
Flavobacterium frigidarium (S3-9)
AY771722
99
0.0
4
Yellow
27
A-7 Ike
Flavobacterium aquatile (DSM1132)
AM230485
97
0.0
4
Orange
28
A-7 Ike
Flavobacterium aquatile (DSM1132)
AM230485
97
0.0
4
Orange
29
B-3 Ike
Psychrobacter cryohalolentis (K5)
CP000323
99
0.0
4
White
30
B-3 Ike
Psychrobacter alimentarius (S3-15)
AY771725
99
0.0
4
White
31
B-1 Ike
Methylobacterium adhaesivum (AR27)
AM040156
99
0.0
4
Dark red
32
A-7 Ike
Flavobacterium aquatile (DSM1132)
AM230485
97
0.0
4
Orange
33
A-6 Ike
Cryobacteriu psychrophilum (DSM4854)
AJ544063
99
0.0
4
Orange
35
Tokkuri Ike
Psychrobacter maritimus (Pi2-20)
AJ609272
99
0.0
4
White
36
Tokkuri Ike
Flavobacterium frigidarium (S3-9)
AY771722
99
0.0
4
Buff
37
Tokkuri Ike
Psychrobacter maritimus (Pi2-20)
AJ609272
99
0.0
4
White
41
Suribati Ike
Psychroflexus torques (BSi20642)
DQ007442
97
0.0
4
Orange, slimy
42
A-7 Ike
Brevundimonas variabilis (ATCC15255)
AJ227783
99
0.0
4
Nigger-brown
43
A-7 Ike
Brevundimonas variabilis (ATCC15255)
AJ227783
99
0.0
4
Sandy beige
44
A-7 Ike
Flavobacterium aquatile (DSM1132)
AM230485
97
0.0
4
Brown
45
A-7 Ike
Blastomonas natatoria (2.3.)
AJ250435
98
0.0
4
Puce
by spreading the Antarctica lake samples directly onto
agar-media plates but twenty strains were isolated at 4 °C by
filtration Antarctica lake samples, it was thought that microorganisms
in Antarctica lakes had been subjected to low temperature and
limiting nutrient for long time, high incubation temperature
and rich nutrient medium might be stressor for Antarctic
microorganisms. Therefore, incubation at low temperature with
media not containing rich nutrition were more suitable for
Antarctica lake microorganisms because the isolated microorganisms
were most likely to be adapted to the often oligotrophic
conditions of many cold habitats. Although the developing
method of dealing with Antarctica lake sample was difficult,
the procedure refracting the Antarctic environment led to the
good result. The concentration of bacterial density with filtration
increased the number of recovered isolates, which indicated
that it was also important factor of concentration of cell
number because the bacterial density in Antarctica lakes
might be very low. In order to isolate microorganisms strictly
and know its temperature adaptability of bacteria incubated at
4 °C, strains were re-streaked. As a result, strain No. 23, 25,
and 26 formed each colony on PYMV agar plates after only 2
days incubation at 20 °C; however, most of other strains
formed each colony on PYMV or R2A agar plates with slower
growth requiring incubation for 1-3 weeks at 20 °C.
According to the definition and category of psychrophiles by
Morita [10], psychrophiles has been further subdivided into
psychrophiles sensu stricto, which have optimal growth
temperature below 15 °C and an upper limit of 20 °C, and
psychorotrphs (psychrotolerants) which are able to divide at 0
°C or below and grow optimally at temperatures around 20-25
°C. It has the possibility that strain No. 23, 25, and 26
belonged to psychorotrphs (psychrotolerants), whereas most
of other strains belonged to psychrophiles sensu stricto.
Characterization based on 16S rDNA sequence
As a result (see Table I) of sequencing and homology
analysis, four clones (strain No. 31, 42, 43, and 45) showed the
high similarity to Alpha-proteobacteria, seven clones (strain
No. 22, 23, 24, 29, 30, 35, and 37) were similar to
Gamma-proteobacteria, one clone (strain No. 33) was similar
to Actinobacteria and one clone (strain No. 20) was similar to
Bacilli. Nine clones (strain No. 15, 25, 26, 27, 28, 32, and 41)
were similar to Fibrobacter. Strain No. 25 had 99%, 100%of16S
rDNA sequence similarity with strain No. 26 and strain No. 36,
separately. Also, strain No. 27 had 100% of 16S rDNA sequence
similarity with strain No. 28 and strain No. 44. Strain No. 42 had
100% of 16S rDNA sequence similarity with strain No. 43.
These results showed that some strains might be similar or
same species. The analysis of the community by obtaining
and comparing isolates not only contributes to the general
knowledge of Antarctic microorganisms but also a large
collection of organisms that can be further characterized and
useful to several fields. Naganuma et al. have isolated some
euryhaline halophilic strains from Suribati Ike [3]. Some strains
have 100% 16S rDNA (about 1465 bp) sequence similarity with
Marinobacter sp. in GenBank Database; however the
physiological characteristics were different such as growth in
different temperature, anaerobic or aerobic, and survival in
different NaCl concentration. Comparison of the genomic and
physiological characteristics among our isolates also might
increase much understanding of the microbial diversity of this
environment.
16S rDNA sequencing results suggested that three of the
isolates may represent new species. Strain No. 15, 41, and 27
(28, 32, and 44) has the highest 93%, 97%, and 97% 16S rDNA
sequence similarity with the GenBank database respectively.
Strain No. 15, 41, and 27 (28, 32, and 44) are likely novel species
in the genus Gillisia, Psychroflexus, and Flavobacterium which
belong to the Flavobacteria group. The potential novel strains
isolated constitute a unique collection for further taxonomic
analysis, physiological characterization and screening. More
studies of these microorganisms in Antarctic lakes may
increase the molecular and biochemical several advantages
such as antifreezing proteins.
From this survey, we overcame the difficulty of recovery
culturable organisms from Antarctica with comparison several
incubation conditions ofthe microorganism such astemperature,
nutrient concentration, andthenatural growth habitat, and
could
get therepresentativemicrobial microorganismsfromlow temperature
environments, proposing the possibility that several useful
microorganisms might be isolated from low temperature zone in
the future using techniques like this study.
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