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3173
Advances in Environmental Biology, 5(10): 3173-3178, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Isolation and characterization of some aquatic bacteria from Qurugöl Lake in
Azerbaijan under aerobic conditions
1,2
Vahideh Tarhriz, 1Ghorbanali Nematzadeh, 3Fatemeh Mohammadzadeh, 1Ehtaram Rahimi,
and 2Mohammad Saeid Hejazi
1
Rice and Citrus Research Institute (RCRI), Agricultural Sciences and Natural Resources University (SANRU),
Sari, Iran.
2
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences,
Tabriz, Iran.
3
Faculty of Agriculture, Zanjan University, Zanjan, Iran.
Vahideh Tarhriz, Fatemeh Mohammadzadeh, Mohammad Saeid Hejazi, Ghorbanali Nematzadeh,
Ehtaram Rahimi: Isolation and characterization of some aquatic bacteria from Qurugöl Lake in
Azerbaijan under aerobic conditions
ABSTRACT
Aquatic bacteria play crucial roles in environmental biology. They are a rich source of new genes;
unfortunately most of them are left unknown and regarding the ones which have been recognized and known
there is not enough and efficient information considering their applications. Considering the geographical
conditions of Qurug l Lake that is mostly frozen during the cold seasons and also lack of published papers on
the presence of aquatic bacteria of this lake, the present study aimed to investigate the bacterial community in
the lake. In so doing, samples were taken from four different parts and at each part they were taken from three
different depths 0.25, 1 and 8 meter, respectively. The taken samples were grown on special media including:
modified marine agar and sea water medium (SWM). Biochemical tests consisted of determination of oxidase
and catalase activity, gelatin liquefaction, ability to hydrolyze starch and casein, production of indole, H2S and
nitrate reduction tests were carried out. Thirty two isolates recognized. Most of isolated strains were Gramnegative (21) and mostly were psychrophilic bacteria. According to the results obtained from the analysis of
extra-cellular enzymes it was found that the isolated bacteria have high variety hydrolytic enzyme activity. In a
way that few of the isolated bacteria could produce a mixture of the mentioned enzymes, but all in all Grampositive bacteria in comparison with Gram-negative bacteria tested showed more hydrolytic activities, also
revealed that the capability of Gram-negative bacteria in production of lipase enzyme is more than Grampositive bacteria. On the basis of phylogenetic results, it is recommended that the isolates belong to Aeromonas,
Alishewanella, Alteromonas, Jeotgalicoccus, Bacillus, Marinobacter, Marinococcus, Paracoccus,
Pseudoalteromonas, Rheinheimera, Rodobacter, Prophyrobacter and Shewanella. Moreover, several members
of the Alishewanella, Jeotgalicoccus, Bacillus, Marinococcus and Shewanella clades were recovered. In a way
that Alishewanella clade was dominant.
Key words: aquatic bacteria, hydrolytic enzymes, Qurug l Lake, 16S rRNA gene.
Introduction
Aquatic microorganisms have very important
roles in food chains, biogeochemical cycling,
decomposition and production industrial enzymes,
yet they are largely uncharacterized due to their
small size, the limited range of morphologies, and
the difficulties in obtaining pure cultures that are
representative of natural populations [16][10]. It is
estimated that negligible percent of aquatic
microorganisms can be recovered through standard
tissue culturing technique. However, the action of
recovery can be improved through applying
molecular phylogenetic analysis such as 16s rRNA
gene sequence [10]. Most of aquatic bacteria are a
source of hydrolytic enzymes such as amylases,
DNases, lipases, proteases, pullulanases and other
important industrial enzymes. For example Vibrio
alginolyticu produces six proteases. One of these
proteases, Pro A, is SDS resistant [5]. Also most of
the metabolites produced by aquatic bacteria
especially Cianobactéria have acute cytotoxic
Corresponding Author
Vahideh Tarhriz, Rice and Citrus Research Institute (RCRI), Faculty of Agriculture, Sari
Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran.
Email: [email protected]; Tel: +98 (411) 337 2256; Mob: +98 914 311 3708
Fax: +98 (411) 334 4798
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Adv. Environ. Biol., 5(10): 3173-3178, 2011
feature such as Apratoxin, private neurotoxic activity
such as Kalkilatoxin and Antillatoxin or anti-cancer
feature such as Dolastatin and Cutacia [17].
Qurugӧl Lake is a fresh water Lake and it is
neglected to be studied for its potential interesting
novel types of aquatic bacteria. Due to importance of
aquatic bacteria, it is the aim of this paper to isolate
and characterize of aquatic bacteria in Qurugӧl Lake
by 16s rRNA gene, since according to our knowledge
such studies have not been carried out up to now in
this lake.
Material and Methods
Water Sample Collections:
Samples from four different areas of the lake and
at three different depths 0.25, 1 and 8 meter,
respectively, were collected at the end of rainy
seasons. Then the samples were transported to the
RCRI (Rice and Citrus Research Institute)
laboratory. Isolation and identification of aquaticbacteria were determined on SWM contained (per
liter): Beef extract, 10.0 g; Peptone, 10.0 g; Agar,
20.0 g; Tap water, 250.0 ml; artificial sea water,
750.0 ml was tested, consist of NaCl, 28.13 g; KCl,
0.77 g; CaCl2 x 2 H2O, 1.6 g; MgCl2 x 6 H2O, 4.8 g;
NaHCO3, 0.11 g; MgSO4 x 7 H2O, 3.5 g; and
modified marine agar contained (per liter): peptone,
5.0 g; yeast extract, 1.0 g; Fe3+-citrate, 0.1 g; NaCl,
(1.2, 2.4, 4.8, 9.75, 19.45 g); MgCl2 (dried), 5.9 g;
Na2SO2, 3.24g; CaCl2, 1.8g; KCl ,0.55g; Na2O3,
0.16g; KBr, 0.08g; SrCl2, 34.0mg H3BO3, 22.0 mg;
Na-silicate, 4.0 mg; NaF, 2.4 mg; (NH4)NO3, 1.6 mg;
Na2HPO4, 8.0 mg, and pH was adjusted to 7.6 ± 0.2.
100 micro liters from the samples were inoculated
onto plates. The cultures were incubated at 25°C for
a couple of weeks. To obtain a pure culture, isolates
were taken by marine broth medium and were stored
at -70°C in marine broth supplemented with 30%
(v/v) Glycerol. Gram staining was performed using
the standard Gram staining method [8] and also
Gram reaction was tested with KOH lysis test
technique [9].
Growth of isolates was investigated at -5, 0, 5,
10, 15, 20, 25, 30, 37, 40, 45 and 50 °C in marine
broth containing (per liter): Bacto peptone, 5.0 g;
Bacto yeast extract, 1.0 g; Fe(III) citrate, 0.1 g; NaCl,
19.45 g; MgCl2 (dried), 5.9 g; Na2SO4, 3.24 g; CaCl2,
1.8 g; KCl, 0.55 g; Na2CO3, 0.16 g; KBr, 0.08 g;
SrCl2, 34.0 mg; H3BO3, 22.0 mg; Na-silicate, 4.0 mg;
NaF, 2.4 mg; (NH4)NO3, 1.6 mg; Na2HPO4, 8.0mg;
with pH 7.2±0.5. In order to determine the optimal
growth temperature, spectrophotometric absorbance
of the samples was measured at 600 nm (OD: 600)
after 48 hours.
The pH tolerance was determined in marine
broth with the pH set from 4 and 12 (using
increments of 1 pH unit).
Growth of isolates was tested at different NaCl
concentrations
in
marine
broth
medium
supplemented with 0 and 1-10% NaCl, at intervals of
0.5% (w/v) NaCl [23].
Biochemical tests consisted of determination of
oxidase and catalase activity, gelatin liquefaction,
ability to hydrolyze starch and casein, production of
indole, H2S, nitrate reduction and motility tests were
carried out according to [12].
Screening of strains for extracellular hydrolytic
activities:
In the next step the capability of the isolated
bacteria was analyzed in producing extra-cellular
amylase enzyme (Cowan et al., 1991), extra-cellular
protease enzyme (Simankova et al., 1994), extracellular lipase enzyme (Simankova et al., 1994),
extra-cellular nuclease enzyme (Onishi et al., 1983),
extra-cellular inulinase enzyme (Allais et al., 1986),
extracellular pectinase enzyme (Marcia et al., 1999),
extra-cellular carboxymethyl cellulose enzyme
(Simankova et al., 1994), extra-cellular pullulanase
enzyme (Murray et al., 1994) and extra-cellular
chitinase enzyme (Shaikh et al., 1993). All tests were
carried out at optimum NaCl concentration of each
isolate.
The 16S rRNA gene sequences:
For genotypic characterization, DNA was
isolated from the isolates according to the method
described by Corbin et al. (2001) with some
modifications. For phylogenetic analysis based on
the 16S rRNA gene sequence, the genes were
amplified using PCR technique in the presence of
forward 16F27 (5' -AGA GTT TGA TCT GGC TCA
G- 3') and reverse 16R1488 (5' -TAC CTT GTT
AGG ACT TCA CC- 3') primers [11].
The amplified fragments were purified using
Roche kit (Germany) and then sequenced by utilizing
forward and reverse and 3 other designed primers;
H400 (5'-GGG TTG TAA AGC ACT TTC AG-3'),
H550 (5'-CCA GTA ATT CCG ATT AAC GC-3')
and H900 (5'-ACT CAA ATG AAT TGA CGG GG3') used for PCR amplification by Macrogen
company (Korea). The 16S rRNA gene reads were
assembled using Chromas pro software and aligned
using the multiple sequence alignment program
CLUSTAL X (version 1.83) (Thompson et al.,
1997). Phylogenetic trees were constructed using
neighbor-joining, method in MEGA version 4
software package [21].
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Adv. Environ. Biol., 5(10): 3173-3178, 2011
Table 1: Comparison of the characteristics of isolated bacteria based on phenotypic features, Taxa: the some aquatic isolates in each genus.
Table 2. Closest known bacteria to various isolates Qurug l Lake based on 16S rRNA gene sequence.
Fig. 1: The comparison of production of extra-cellular enzymes in Gram-negative and Gram-positive bacteria
existing in Qurug l Lake.
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Adv. Environ. Biol., 5(10): 3173-3178, 2011
Fig. 2: Phylogenetic analysis of the isolates was members of thirteen genera of the domain Bacteria based on a
partial sequence of the 16S ribosomal RNA gene. The sequence alignment was performed using the
CLUSTAL-X program and the tree was generated using the NJ method in MEGA 4 software.
Results and Discussion
We could separate thirty two isolates from
twelve water samples collected from Qurug l Lake,
most of which were Gram-negative (21 isolates).
Colony pigmentation from these samples was red to
white, most of which were cream. All isolates were
catalase positive and some of which were able to
grow in the absence of NaCl (Table 2).
Based on the results obtained from the analysis
of extra-cellular enzymes it was found that the
isolated bacteria have high variety hydrolytic enzyme
activity. In a way that few of the isolated bacteria
could produce a mixture of the mentioned enzymes,
but Gram-positive bacteria in comparison with
Gram-negative bacteria tested showed more
hydrolytic activities. The findings also revealed that
the capability of Gram-negative bacteria in
production of lipase enzyme is more than Grampositive bacteria (Fig. 1).
The comparison of isolated bacteria based on
variety and production of hydrolytic enzymes shows
that Gram-positive bacilli are at the top of the list.
Most of the isolated bacteria in this research have
had the capability to produce DNase enzyme. The
dominant enzyme in Gram-negative was lipase and
Gram-positive was DNase.
In order to find out their phylogenetic position,
the 16S rRNA gene sequence of each strain was
analyzed, and a phylogenetic tree was constructed
(Fig. 2). We were able to sequence the isolates
successfully and the results showed that the
collections have been put into at least thirteen
bacterial genera. The bacterial isolates were mostly
ascribable to members of Aeromonas, Alishewanella,
Alteromonas,
Jeotgalicoccus,
Bacillus,
Marinobacter,
Marinococcus,
Paracoccus,
Pseudoalteromonas, Rheinheimera, Rodobacter,
Prophyrobacter and Shewanella. Moreover, several
members of the Alishewanella, Jeotgalicoccus,
Bacillus, Marinococcus and Shewanella clades were
recovered. In a way that Alishewanella clade was
dominant.
Discussion:
In the last few years, increased attention has
been given to aquatic bacteria, especially producing
hydrolytic enzymes bacteria. Several studies have
been conducted on their biotechnological
applications as well as their ecology and phylogeny
[18]. There are some studies about aquatic bacterial
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Adv. Environ. Biol., 5(10): 3173-3178, 2011
communities of several lakes by amplification and
sequencing of 16S rRNA gene such as study of
aquatic bacteria communities in the Adirondack
Mountains lakes by [10].
The present study tries to make an attempt to
study the isolation and characterization of some
aquatic bacteria from Qurugöl Lake under aerobic
condition. This study, as far as we know, provides
the first publication on the diversity of aquatic
bacteria in Qurugöl Lake in Azerbijan-Iran.
Even though from morphological point of view
most of the separated isolates are cold environment
favoring kind due to their ability to grow in less than
five centigrade, the best temperature for the growth
of the isolates was determined as between 25 to 30
ºC (Table 1). Furthermore the isolates were able to
grow in a wide range of pH, even though the ideal
conditions for the growth of majority of them is
determined in pH=6.5-7.
Throughout the course of this work, we isolated
thirty two strains. Most of the strains grow in the
absence of NaCl. Phylogenetic analysis indicated
that all isolates were members of thirteen genera of
the domain bacteria, in five of which were dominant;
Alishewanella,
Bacillus,
Jeotgalicoccus,
Marinococcus and Shewanella. The collection
contains only one member from each of the
Alteromonas, Pesodoalteromonas, Porphyrobacter
and Rhodobacter(Table 2).
Six strains which were isolated on marine
medium belonged to Alishewanella genus, most of
which had high level of 16S rRNA gene similarity
with Alishewanella aestuarii (>99%) (Table 1), but
Similarities between strain RCRI4 (=RCRI16)
(=BCCM/ LMG 26473T = JCM 17275T) and closely
related type strains, achieved using the EzTaxon
(Chun et al., 2007), were 98% with Alishewanella
agri BLO6T. So the GenBank/EMBL accession
number for the 16S rRNA gene sequences of strain
RCRI4 (=RCRI16) is GQ505294. These strains are
Gram-negative, rod shaped, nonmotile with 1.5-2.0
μm long, and 0.9 μm width, the biochemical tests
indicated that the strain RCRI4 is capable to produce
amylase, lipase, DNase, inulinase, pectinase, but is
not capable to produce other hydrolytic enzymes.
The phylogenetic tree showed that strains
RCRI7 (=RCRI8) and RCRI18 (= JCM 17276T) fall
within the radiation of a cluster composed of
Shewanella species ≥99.1%. 16S rRNA gene
sequence similarity with Shewanella xiamenensis,
Shewanella putrefaciens and Shewanella oneidensis
(Fig. 2; Table 2). They could produce lipase and
amylas, but they were not able to produce other
hydrolytic enzymes. The GenBank/EMBL accession
number for the 16S rRNA gene sequences of strain
RCRI7 (=RCRI8) is GQ988720.
Also the closest genus with strain RCRI19
(=BCCM/ LMG 25773T =JCM 17277T) is genus
Rhodobacter spp. Phylogenic tree clustered this
strain with Rhodobacter blasticus(95.3%). The strain
is Gram-negative, rod shaped and motile with 1.3-3.0
μm length, and 0.9 μm widths. The strains grow in
the absence of NaCl and also in the presence of 0.5
and 1 percent NaCl, but not in 1.5% NaCl in marine
agar. The strain RCRI19 was able to produce only
lipase and amylase. The 16S rRNA gene sequences
of strain RCRI19 was scored HQ392507 accession
number in the GenBank/EMBL.
Conclusion:
Among the culture collection strains tested for
hydrolytic enzyme activities most of them were
negative for the production of the enzymes, only two
genera Bacillus and Marinococcus showed the
highest variety of production of hydrolytic enzyme
activities (Table 1), both of them belong to Grampositive bacteria. Since dominant bacteria (work in
progress) were Gram-negative, so this may be
influence the results. In comparison with Gramnegative species, the Gram-positive bacteria tested
showed more hydrolytic activities, particularly
DNase enzyme production. Between the Grampositive, representatives of the genus Bacillus was
predominant. The genus Bacillus is well known as an
enzyme-producer and many industrial processes use
species belonging to this genus for commercial
production of enzymes (Sanchez-porro et al., 2003).
Finally xylanase producers are limited to
representatives of the genus Bacillus.
In exhaustive comparison with moderately
halophilic bacteria, it was sound fresh water bacteria
with less tolerant NaCl, were very poor to produce
hydrolytic enzyme activities, in the other hands
halophile and halotolerant bacteria were higher
variety producers of hydrolytic enzymes.
Furthermore, the potential use of these
microorganisms in degrading aromatic compounds as
underlined earlier (Dyksterhouse et al., 1995),
prompts us to screen our collection of aquatic
bacteria for their ability (work in progress). Besides,
it is reported that marine bacteria Marinobacter,
jeotgalicoccus and Bacillus strains, are able to
degrade organic pollutants [7]. Study of our
collection for enzyme production of these bacteria to
degrade environmental pollutants, is another step in
our interests.
Acknowledgment
We highly appreciate the Rice and Citrus
Research institute (RCRI) for financial supporting
also, Pharmaceutical Biotechnology Department,
Faculty of Pharmacy, Tabriz University of Medical
Sciences.
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