Download Advances in Environmental Biology

Advances in Environmental Biology, 8(13) August 2014, Pages: 540-543
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Isolation and Identification of New Species of Thermophilic Cyanobacteria from
Hot Springs in Northern Iran, and Investigating its Ability to Remove Heavy
Metals
1Mohamadi
1
2
3
Ebli F, 2Heshmatipour Z, 3Khanafari A, 1Aslikousha H
MS.c in Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
Faculty Members, Department of Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
Department of Microbiology, Tehran North Branch, Islamic Azad University, Tehran, Iran
ARTICLE INFO
Article history:
Received 25 April 2014
Received in revised form
8 May 2014
Accepted 20 July 2014
Available online 18 August 2014
Keywords:
Hot springs in northern Iran, BG11,
cyano EBLI1
ABSTRACT
A large family of bacteria are cyanobacteria, which have a large variety of types and
been found in various habitats. Heavy metals such as copper, lead, and zinc−as a large
group of elements considered as toxic factors−present in such environment as waste
water and soil. Some bacteria, cyanobacteria for example, have the ability to remove
heavy metals. The purpose of this study is isolation and identification of new species of
thermophilic Cyanobacteria and investigating its ability to remove heavy metals. First,
the collected samples were inoculated into BG11 broth medium. Then the mass was
transferred to the BG11 agar medium and were incubated for 8-10 days. Finally,
molecular diagnosis 16s RNA was developed on the colonies. thermophilic
Cyanobacteria were inoculated in medium containing copper, lead, and zinc; results
were calculated by atomic adsorption. In this study, we succeeded in isolating
unculturable of thermophilic cyanobacteria EBLI1 (89.2%) that was morphologically
similar to oscillatoria and lyngbia. Cyanobacteri have also been identified as capable of
elimination of 2.5% zinc, 13.5% copper, and 27% lead from the media. The study has
proved that not only heavy metals such as zinc, copper, and lead are not toxic for
cyanobacteria, but they can reduce GT time bacteria.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Mohamadi Ebli F, Heshmatipour Z, Khanafari A, Aslikousha H., Isolation and Identification of New Species of
Thermophilic Cyanobacteria from Hot Springs in Northern Iran, and Investigating its Ability to Remove Heavy Metals. Adv. Environ. Biol.,
8(13), 540-543, 2014
INTRODUCTION
Cyanobacteria are the microorganisms which exist in any places. Cyanobacteria are also known as bluegreen algae, blue-green bacteria. Thermophilic Cyanobacteria are interesting study organisms for basic as well
as for applied research. Their ancestors are possibly the oldest primary producer organisms common in the
distant past, and they perhaps used thermal springs as refugia [1]. Have recently compared 37 strains of
Mastigocladus laminosus isolated from sites throughout the world hexadecenoic acids have a possibly important
role in the adaptation of Cyanobacteria to high temperatures and the ratio between saturated and unsaturated
fatty acids (s/u ratio) decreases with decreasing temperature of a thermophilic strain of Synechococcus [2]. In
mesophilic strain cultivated at 25-32 0c the s/u ratio was increased. A change in the s/u ratio, the mechanism of
adaptation of algae to high temperature, was documented from a quite opposite spectrum of conditions.
Cryoseston species of Chloromonas, isolated from Antarctica, had a higher proportion of unsaturated acids,
enabling the functioning of cells in temperatures just above zero [3]. Thermal springs represent pools of new
strains possessing attractive biochemical pathways and unusual metabolic product for biotechnological
applications. For example, the thermo tolerant Phormidium sp. produced an anti-microbial material against G,G+ bacteria, Candida albicans and Cladosporidium resinae [4]. Another Phormidium sp., immobilized in
calcium alginate, was used for treatment of dye-rich wastewater [5]. Cancer drugs were produced from
thermophilic Cyanobacteria by javor [6]. Some unusual Fe-proteins, siderophores, were identified in some
thermophiles [7]. Ferredioxins from Mastigocladus laminosus, however, act as toxin and restrict public use
exploitation of a spring in saudi Arabia [8]. Thermophilic Cynechococcus sp. is a potential producer of poly-Bhydroxy butyrate,which is the basis of biologically degradable plastics [9]. Production of hydrogen by some
Corresponding Author: Mohamadi Ebli F, MS.c in Microbiology, Tonekabon Branch, Islamic Azad University,
Tonekabon, Iran,
E-mail: [email protected]
541
Mohamadi Ebli F, et al., 2014
Advances in Environmental Biology, 8(13) August 2014, Pages: 540-543
Cyanobacteria is a promising source of energy for the future [10]. The exploitation of natural hot water with a
large content of co2 is highly profitable for algal biotechnology, e.g. production of Sprulina [11] and potentially
for production of thermal species. The purpose of this study is isolation and identification of new species of
thermophilic Cyanobacteria and investigating its ability to remove heavy metals such as: lead, copper and zinc.
MATERIAL AND METHOD
Sampling:
The Samples were prepared from thermal hot springs from the depth ranging 30 to 50 cm, under standard
condition for 3 times, 15 samples in each time. The samples were transferred to the lab at the same thermal
spring temperature-without decreasing it-in pyrex glass containers and high quality flasks, in order to preserve
the same temperature.
Cultivation in broth medium:
The samples were inoculated into selective broth medium of thermophilic Cyanobacteria. Media prepared
in this research include BG-11 broth medium and DG castenholz broth medium. The samples were then
incubated at 500c for 6 to 7 days, at 8-10% of co2 and 150 rpm.
Cultivation in solid medium:
100 to 200 λ from broth medium was transported on to plate .due to high temperature of incubator and
possible drying out of the plates ,first we covered all over the plates with parafilm and put them into a zip pack.
At the end we used wet cotton balls in order to provide the optimum humidity of the plates. Finally the plates
were incubated at 550c, 8-10% co2 for 8 to 10 days.
Investigation of morphological characteristics using fluorescence and light microscopes:
In order to observe Cyanobacteria microscopically, we used fluorescence and light microscopes. For
observation, first a drop of strile water on slides and then some of colonies made in solid medium were laid on
slide using lamella. They were impressed by a slide and this prepared slide examined under the microscopes.
Molecular diagnosis:
In this step the created colonies were tested for identification and final confirmation. Molecular techniques
were also used and DNA extraction using phenol-chloroform method and the bacteria were identified by 16s r
DNA.
PCR Reaction:
Multiplication was carried out using 2 special primers of forward and reverse. The primers include:
CYA106F
(5ʹ-CGGACGGGTGAGTAACGCGTGA-3ʹ)
and
CYA781R
(5GACTACWGGGGTATCTAATCCCWTT -3ʹ).
Elimination of heavy metals:
A certain amount of Thermophilic Cyanobacteria was cultivated in broth medium containing a certain
amount of heavy metals such as zinc, lead and copper and incubation was carried out under condition at 550c,
150 rpm and 3000 lux of light for 4 days.
RESULTS AND DISCUSSION
Results:
The initial investigation, when we were observing the isolated bacteria under light (Fig. 1) and fluorescence
microscopes (Fig. 2), we observed that Cyanobacteria were so similer to bacteria orders Oscillatoria, genus
Oscillatoria and Lyngbia. The isolated bacteria were morphologically and physically similar to the genus, but
after molecular diagnosis and using the special primers, the result showed that the bacteria belonged to strain
EBLI1
Morphological characterization of cyano EBLI1:
1: Trichomes straight or loosely coiled for portion of their length
2: Cells disk-shaped (wider then long)
3: Cells isodiametric or cylindrical
4: Trichomes with distinct sheat
5: Trichomes without or with very thin sheath
542
Mohamadi Ebli F, et al., 2014
Advances in Environmental Biology, 8(13) August 2014, Pages: 540-543
Fig. 1: Cyano EBLI1 by light microscopy.
Fig. 2: Cyano EBLI1 by fluorescence microscopy.
The result of atomic adsorption:
In the next investigation it was confirmed that the Thermophilic Cyanobacteria were able to eliminate
heavy metals. Measuring the amount of heavy metals by atomic adsorption confirmed that the Thermophilic
Cyanobacteria were able to remove 2.5% zinc, 13.5% copper, and 27% lead from the media.
Table 1: The result of atomic adsorption.
Metal
Cyanobacteria+ Media
Cyanobacteria+Media+Heavy Metal
Heavy Metal+media
Zn
0/019 mg/l
0/023 mg/l
0/035 mg/l
Pb
0/20 mg/l
0/29 mg/l
0/38 mg/l
Cu
0/104 mg/l
0/210 mg/l
0/320 mg/l
Discussion:
In 1807, olrich, a German researcher was able to isolate Thermophilic Cyanobacteria from hot spring
zerkamein in German. ThisCyanobacteria belonged to order Synechococcus [12]. Buchholz gave a exactly
microscopical description of a green substance existed in these hot springs in 1912 [13]. popke and partners was
able to isolate two Thermophilic Cyanobacteria called: Synechococcus and Oscillatoria in Izmir from turkey
(balcova region) [14]. Their result was so much similar to ours from under taken studies. For isolating popke
utilized castenholz DG broth medium which had no answer for us in this research. Therefore, we used BG-11
broth medium for isolating in our studies. We imagined that heavy metals-that they often exist as a factor in
environments inhibit the growth of bacteria and work as inhibitors. But the result from our studies showed that
not only the metals such as lead, zinc and copper, are not inhibitors but they work as growth factors. They also
cause generation time of Thermophilic Cyanobacteria to decrease. They reduce their generation time from 15
days to 7 days. In this study the result showed that some heavy metals such as lead, zinc and copper in low
amount, are necessary for the growth of Thermophilic Cyanobacteria and work as growth factors. However, it’s
possible that high amount of them can be inhibitor growth.
543
Mohamadi Ebli F, et al., 2014
Advances in Environmental Biology, 8(13) August 2014, Pages: 540-543
Anabaena sp. strain 90.
Anabaena cylindrica PCC 7122 strain CCAP 1403/2B.
Anabaena bergii strain AB2010/08.
Anabaena azotica
Anabaena variabilis
Oscillatoria prolifera
Oscillatoria amoena CCAP 1459/39
Cyanobacterium sp. POKH2
Uncultured Cyanobacterium sp. clone ATLA Crab Bac C07.
Oscillatoria sancta
Oscillatoria sp. zan1mo51
Oscillatoria duplisecta ETS-06
Uncultured Oscillatoria sp. clone GMMC 16S 81.
Oscillatoria acuminata
Cyanobacterium sp. CENA169
Cyanobacterium sp. PBJ2
Cyanobacterium ebli strain ebli
Synechococcus sp. (strain NIVA-CYA 328).
Synechococcus elongatus PCC 6301 strain CCAP 1405/1.
Cyanobacterium sp. THH
Cyanobacterium stanieri PCC 7202
Cyanobacterium sp. CENA527
Cyanobacterium sp. MBIC10216
Cyanobacterium aponinum ETS-03
Cyanobacterium sp. Oman I
Uncultured Synechococcus sp. clone GMMC 16S 66.
Oscillatoria tenuis NIES-33
Synechococcus sp. strain PCC 7002.
Cyanobacterium sp. MT
Oscillatoria sp. CCMEE 416
Fig. 3: Phylogenetic tree of new thermophilic cyanobacteria (cyano ebli1).
REFERENCE
[1] Adhikary, S.P., 2006. Blue green algae, survival strategies in diverse environmemnt. Pointer Publishers,
Jaipur (Raj) India.
[2] Anagnostidis, K., 1961. Untersuchungen über die Cyanophyceen einiger Thermen in Greichland. PhD
Thesis, University of Thessaloniki.
[3] Anagnostidis, K., J. Komrek, 1990. Modern approach to the classification system of Cyanopytes, 5,
Stigonematales. Algological Studies, 59: 1-73.
[4] Becher, P.G., H.I. Bumann, K. Gademann, 2009. The cyanobacterial alkaloid nostocarboline: an inhibitor
of acetylcholinesterase and trypsin. Journal of Applied Phycology, 21: 103-110.
[5] Bhandari, R., P.K. Sharma, 2006. High-light-induced changes on photosynthesis, pigments, sugars, lipids
and antioxidant enzymes in freshwater (Nostoc spongiaeforme) andmarine (Phormidium corium)
cyanobacteria. Photochemistry and Photobiology, 82: 702-710.
[6] Bhandari, R., P.K. Sharma, 2007. Effect of UV-B and high visual radiation on photosynthesis in freshwater
(Nostoc spongiaeforme) and marine (Phormidium corium) cyanobacteria. Indian Journal of Biochemistry
and Biophysics, 44: 231-239.
[7] Bidigare, R.R., M.E. Ondrusek, R. Iturriaga, H.R. Harvey, R.W. Hoham, S.A. Macko, 1993. Evidence for a
photoprotective function for secondary carotenoids of snow algae. Journal of Phycology, 29: 427-434.
[8] Bryanskaya, A.V., V.K. Orleanskii, O.P. Dagurova, 2008. Alaboratory model of the cyanobacterial mat
from Kotel´nikovskii hot spring (Baikal region). Microbiology, 77: 490-496.
[9] Castenholz, R.W., 1969. Thermophilic blue-green algae and the thermal environment. Bacteriological
Reviews, 33: 476-504.
[10] Castenholz, R.W., 1972. The occurrence of the thermophilic blue-green alga, Mastigocladus laminosus, on
Surtsey in 1970. Surtsey Progress Report, 6: 14-19.
[11] Castenholz, R.W., 1973. The possible photosynthetic use of sulfide by the filamentous phototrophic
bacteria of hot springs. Limnology and Oceanography 18: 863-876.
[12] Oren, A., B.J. Tindall, 2005. Nomenclature of the cyanophyta/ cyanobacteria/cyanoprokaryotes under the
International Code of nomenclature of Prokaryotes. Algological Studies (Cyanobacterial Research 6) 117:
39–52.
[13] Buchholz-Cleven, B.E., E.B. Ratunde, K.L. Straub, 1997. Screening for genetic diversity of isolates of
anaerobic Fe(II)-oxidizing bacteria using DGGE and whole-cell hybridization. Syst Appl Microbiol, 20:
301–309.
[14] Papke, R.T., N.B. Ramsing, M.M. Bateson and D.M. Ward, 2003. Geographic isolation in thermophilic
cyanobacteria. Environ. Microbiol, 5: 650-659.