جداسازی و شناسایی مقدماتی ریزجلبک‮ها و سیانوباکترهای بومی لاگون‮های نفتی جزیره خارک‬‬‬‬‬‬‬‬‬‬‬‬‬‬

نوع مقاله: پژوهشی- انگلیسی

نویسندگان

1 استادیار بیوتکنولوژی دانشگاه الزهرا (س) و مرکز ملی ذخایر زیستی و ژنتیکی ایران

2 دانشیار میکروبیولوژی دانشگاه تهران، مرکز ملی ذخایر زیستی و ژنتیکی ایران

3 دانش آموخته کارشناسی ارشد میکروبیولوژی مرکز ملی ذخایر زیستی و ژنتیکی ایران

4 دانشیار ژنتیک ملکولی دانشگاه علم و فرهنگ، و مرکز ملی ذخایر زیستی و ژنتیکی ایران

5 استاد گیاه شناسی دانشگاه پیام نور، ایران

چکیده

مقدمه: جلبکها به لحاظ اکولوژیکی، تنوع زیستی، کشاورزی، پزشکی، زیست فناوری، صنعت و سایر، بسیار مهم هستند. این موجودات توانایی زیادی در تولید زیست مواد فعال، زیست پالایی و تولیدات اولیه دارند. بنابراین، دستیابی به سویههای بومی ایران که دارای تواناییهای بیوتکنولوژیکی و اکولوژیکی دارای اهمیت است.
مواد و روش‏‏ها: آب و خاک منطقه از ایستگاه‌های مختلف جزیره خارک (خلیج فارس) جمع آوری شدند. نمونه‌ها کشت و به کمک روش‌های مختلف خالص‌سازی شد. برای خالص‌سازی از 7 آنتی‌بیوتیک و سایر روش‌های فیزیکی استفاده شد.
نتایج: در این پروژه 7 سویه شامل 5 سیانوباکتر و 2 ریز جلبک کشت و خالص‌سازی شده است. آنتی‌بیوتیک‌های ایمیپنم و سیکلوهگزامید به عنوان بهترین پادزی‌ها برای خالص‌سازی کشت‌ها استفاده شدند. سه سویه به دست آمده به لحاظ ریخت شناسی در عین شباهت به سویه‌های Anabaenopsis، Pseudanabaena teremula،
 Arthronema afaricanum، تفاوت‌هایی هم با آنها دارند که به کمک کلیدهای ریخت شناسی امکان تعیین قطعی گونه وجود ندارد (این گونه ها Kh.T.2، Kh.T.1 و Kh.C.D2 نام گذاری شده اند).
بحث و نتیجهگیری: بر اساس نتایج بدست آمده و شواهد و صفات ریخت‌شناسی، سویه‌های جدا شده در سطح جنس شناسایی شده‌اند و دارای صفات حدواسط هستند. بنابراین، آزمون‌های تکمیلی مثل نشانگرهای ملکولی ITS و 18s rRNA  و 16s rRNA و تعیین توالی ژن‌ها می‌تواند در تایید گونه‌ها مفید باشند.در صورت تایید سویه‌های جدید تبیین صفات ریخت‌شناختی برای آنها شناسایی آسان آنها ضروری است. بهترین آنتی‌بیوتیک برای خالص‌سازی جلبک‌های یوکاریوت، ایمیپنم و برای سیانوباکترها، سیکلوهگزامید است.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

The isolation and preliminary characterization of native cyanobacterial and microalgal strains from lagoons contaminated with petroleum oil in Khark Island

نویسندگان [English]

  • Mostafa Noroozi 1
  • Mohammad Ali Amozegar 2
  • Rezvan Rahimi 3
  • Seyed Abolhassan Shahzadeh Fazeli 4
  • Gholamreza Bakhshi Khaniki 5
1 Assistant Professor of Biotechnology, Alzahra University, Tehran, Iran
2 Associate Professor of Microbiology, University of Tehran and Iranian Biological Resource Centre (IBRC), Iran
3 M.Sc. of Microbiology, Iranian Biological Resource Centre (IBRC), Tehran, Iran
4 Associate Professor of Molecular Genetics, University of Science and Culture, and Iranian Biological Resource Centre (IBRC), Tehran, Iran
5 Professor of plant biology Payame Noor University, Tehran, Iran
چکیده [English]

Introduction: Algae has many applications in terms of ecology, biodiversity, agriculture, medicine, biotechnology, industry, etc. They are potent organisms in bio-active compound production, bioremediation and primary producer. Therefore, it is important to discover local strains with biotechnological and ecological applications.
Materials and methods: Soil and water samples were collected from different sites of Khark Island (Persian Gulf). The samples were cultivated and purified using different techniques. Seven different antibiotics together with other physical methods used to purify the isolates.
Results: Throughout the project 7 strains including 2 eukaryotic algae and 5 cyanobacteria have been isolated. Imipenem and cycloheximide were the best antibiotics for purification of cultures. Three of isolates were morphologically similar to Arthronema africanum, Pseudanabaena teremula, Anabaenopsis sp. However, they have some different characteristics which according to the present identification keys it is not possible to identify their identity (they have nominated Kh.C.d2, Kh.T.1 and Kh.T.2).
Discussion and conclusion: According to the results, isolated strains were identified at the genus level based on morphology characters; therefore the complementary examinations such as molecular identification, ITS, 18s rRNA, 16s rRNA and sequencing can help to approve the strains identity. Upon approval of the new strains account for morphological traits are necessary for their easy identification. The Imipenem antibiotic is the best for eukaryotic algae purification and Cycloheximide is suitable for prokaryotic algae (cyanobacteria) purification.

کلیدواژه‌ها [English]

  • : Cyanobacteria
  • Culture
  • Khark Island
  • Microalgae
  • New species
  • Petroleum pollution
  • Purification

Introduction

The information contained in the algal flora of Iran and Iranian coastal areas, especially in the Persian Gulf and its oil polluted reserves are limited and require more dedication to identify potential genetic (1- 7). A checklist of macroalgae in Persian Gulf and Oman Sea including Kharg Island has been published (8). The algae have many applications in different fields, secondary metabolites, fishery and veterinary food, medicine, agriculture and wastewater treatment. All of the organisms have important rules in our ecosystem and it is necessary to manage a scientific and economic program for their protection and utilization.

Oil leaching from the ships, Oil wells and offshore installations to the Persian Gulf has made many environmental damages. The precipitation of oil carbohydrates and other contaminants will destruct the precipitate ecosystem and deplete the dissolved oxygen, make acidic pH and releasing the heavy metals and toxins to the water. The photosynthesis of Dunaliella was inhibited primarily in contact with petrulium oil spilled from Prestige oil tanker in Spain, however gradually during mutations the cells become resistant to contamination (9).

Algae and plants are able to eliminate petrulium oil contaminations in soil and water through Phytoextraction or phytoaccumulation process (10). Cyanobacteria use for bioremediation to purify different environmental contaminations including oil, sewage, and other pollutants. It is important to identify and understand the algal flora of the polluted area to plan the bioremediation researches. Meanwhile, it is necessary to purify and maintain the local algal species for different purposes especially to provide for future studies.

In this systematic project, some of the algal species from the contaminated and non-contaminated lagoons to petrulium oil has been cultured and identified. The isolated axenic cultures maintained in Iranian Biological Resource Center (IBRC) for long-term preservation.

 

Materials and methods

Khark Island is a coral island with 32Km2 situated in 54 km distance from North West of Bushehr port in the latitude and the longitude of 12°, 29', 30'' N and 50°, 12', 45'' to 50°, 20', 10'' E (11). In coordination with companies and offshore oil terminals, two consecutive samplings were done from sewage lagoon and clean wetland (Baghe Pire Mard) from February 2012 to June 2013.

Sampling Stations: The geographical location and condition of stations have depicted in (fig. 1 and table 1). In the Baghe Pire Mard wetland, no oil contamination were seen and the plants were grown in a good situation.

 

Table 1- Geographical and pollution conditions of the sampling stations

Sampling station

Geographical Location

Contamination condition

Oil Lagoon A

N2922/6 33/5, E5030 29/1

A little contamination with plants growth

Oil Lagoon C

N 29 13 16.5, E 050 19 41.3

Water and soil contaminated to oil

Coastal area in south west of island

N29 21/9 29/8, E50 30/3 24/2

Extreme contamination

Baghe Pire Mard wetland

E050 15 57/5, N29 15 57/5

No oil contamination

 

 

     

South east coastal area

Oil Lagoon A

Oil Lagoon A

Fig. 1- Sampling stations of Khark Island contaminated with oil compounds

 

 

Fig. 2- The growth chamber for photosynthetic organisms

 

 

The water and soil samples were collected in screw cap bottles and kept in 4ºC and transferred to the lab as soon as possible and cultured immediately in BBM, BG11, and Chu (12- 14) medium. Based on table 2, different antibiotics (prokaryotic and eukaryotic) were used to inhibit the growth of undesired organisms. Antibiotic solutions (50 µg/ml) were sterilized with 0.2 µ Milipore filter.

 

Table 2- Utilized antibiotics and their solvents

Antibiotic

Solvent

Ampicillin

Distilled water

Penicillin

Ethanol + DMSO

Cephalexin

Distilled water

Chloramphenicol

Physiologic serum

Imipenem

Ethanol

Nistatin

Water and alcohol

Cycloheximide

Distilled water

The cultured petri dishes were incubated in 16-8 hour’s light-dark period at 25±2 and 2500 to 3000 lux light intensity (fig. 2).

 

Purification Process: According to Richmond different algological and microbiological techniques such as antibiotic, serial dilution, centrifuge, subculture with stereomicroscope and UV irradiation were used to eliminate undesired microorganisms (15). The antibiotics used in the media for purification are depicted in table 2. Cyanobacteria purification is difficult because they grow slowly and have symbiosis and hidden bacteria in their sheath. However, they grow and form a long filament with the ability to move which help to isolate and purify them by a stereomicroscope. The isolated microalgae were cultured in liquid medium and shake manually every day. The growth of cells was determined with OD of the liquid culture with a spectrophotometer and counting the cells with Neubauer slide.

A part of sample fixed with formaldehyde 37% for bright field and phase contrast pictures with Olympus BX51 microscope. The morphologichal charcters such as chloroplast shape,cell size, flagella, unicellular, filamentous or colony, akinet, heterocyst and its position, sheath, form of apical cell, cell borders and many others were used to identify. The following morphological key books were used to identify their species; (16- 19).

 

Results

Based on the physic-chemical characters of the stations (table 3), oil lagoon A and south-west coastline were the most oil contaminated stations and Bghe Pire Mard had no oil pollution. The pH of all stations was alkaline and other chemical parameters are shown in Table 3.

The most effective antibiotic in eliminating bacterial contamination was imipenem (betalactame wide spectrum antibiotics that inhibit the synthesis of peptidoglycans of the cell wall) which is depicted in Table 4. Based on this table and according to Zehnder & Hughes (20) the cycloheximide is against eukaryotic cells (protein synthesis inhibition) and is suitable for cyanobacteria purification, however it weakens the eukaryotic algal growth. The efficiency of penicilin and ampiciline is low due to bacterial resistance. The chloramphenicol and cephalexin were active against cyanobacteria and useful for eukaryotic algal isolation.

The microalgae especially eukaryotic growth was inhibited by increasing antibiotic concentration from 50 to 100 µg/ml. The use of multiple antibiotics in the culture medium can be a toxic effect on microalgae, especially cyanobacteria, and they will lead to growth inhibition and death. Therefore, because of inhibition effect of high dose or multiple antibiotics, it is better to use broad spectrum antibiotic effective on more bacterial group. Resistance and sensitivity of microalgae to various antibiotics has been shown in Table 4.

Nystatin with antifungal activity was not appropriate in this study because of inhibition of the growth microalgae (both cyanobacteria and eukaryotic algae) except in one case (species KH.T.1). In this study 7 strains (5 cyanobacteria and 2 green algae) have been identified and cultured axenically.

 

 

 

Table 3- The physicochemical and petroleum compounds in the samples

Sampling area

TemperatureC°

pH

Ca2+

Mg2+

PO42-

NaCl

Petroleum Compounds

Oil Lagoon C

25

8.2

720

170

0.5

18105

12

Peripheral of oil Lagoon C

25

8

800

194

0.5

18637

16

Oil Lagoon A

25

8

712

211

0.8

16862

14

Peripheral of oil Lagoon A

15

8

720

170

0.9

17217

18

Coastal area south west of island

26

8.3

-

-

-

-

>18

Baghe Pire Mard wetland

25

8.3

811

185

0.9

16316

-

Values is mg/l

Table 4- The sensitivity of isolated algae to antibiotics used in media

Nystatin

Cycloheximide

Imipenem

Chloromphenicol

Cephalexin

Penicillin

Ampicillin

Strain

-

+

+

-

-

+

+

Leptolyngbya nodulosa

-

+

-

-

-

+

+

Strain Kh.C.d2

-

+

-

-

-

+

+

Cyanobacterium aponinum

+

+

+

-

-

+

+

Strain KH.T.1

-

+

+

-

-

+

+

Strain KH.T.2

-

-

+

+

+

+

+

Chlorella sorokiniana

-

-

+

+

+

+

+

Chlorococcum ellipsoideum

(+)growth (-)lack of growth

 

     

Chlorella sorokiniana

1000X

Chlorococcum sp.

2000X

Cyanobacterium aponinum

1000X

     

Leptolyngbya nodulosa

2000X

Kh.c.d2 (Anabaenopsis abijatae)

2000X

Kh.T.1 (Arthronema africanum)

2000X

   

 

Kh.T.2 (Pseudoanaba sp.)

1000X

Kh.T.2 (Pseudoanaba sp.)

2000X

 

Fig. 3- Photomicrograph of identified and cultured strains from Khark Island.

 

The photomicrograph at light microscopy level of isolated species is shown in fig. 3. Four species of cyanobacteria are filamentous which three of them have not been identified exactly because of their new characters which have not been mentioned in the identification keys.

Species Kh.C.D1 with 2 to 4 µm terminal heterocyst and cylindrical and barrels shape akinet (similar to Anabaenopsis abijatae).

The specie Kh.T.1 contain distinct gelatinous sheath around the trichome and regular cells (similar to Arthronema africanum).

Species Kh.T.2 had gelatinous sheath around the trichome and polymorphism shape with some short and disk cells (similar to Pseudanabaena teremula).

One of the cyanobacteria (Cyanobacterium aponinum) and two eukaryote algae were unicellular shown in fig. 3.

Discussion and conclusion

In the present study, the media BBM, BG11, and Chu No.10 were used for cultivation and purification of microalgae which their effectiveness demonstrated in previous studies (21, 22). Types of media can influence the rate of growth, biomass, cell shape, trichome, pigments and even the morphology of algae. The results showed that, compared with two other media, BG11 was suitable for the growth of cyanobacteria and BBM and Chu No.10 medium for the growth of eukaryotic algae.

The basic problems of cultivation and preparation of algal biomass are existence of a variety of organisms and contamination which deleting any one of them requires its own procedures. Based on the obtained results imipenem inhibit bacteria and cycloheximide inhibits the growth of fungi and molds which is appropriate for cyanobacterial cultures. As a conclusion, these two antibiotics recommend for algal and cyanobacterial culture purifications.

Among the oil polluted sampling areas, oil lagoons C with the lowest pollution, and Baghe Pire Mard without oil pollution had the highest number of isolates and the coastal region of South West Khark Island with the highest level of pollution, had the lowest number of isolates. Our study has confirmed the previous researches showing that high concentrations of oil pollutants can cause deactivation of critical processes such as photosynthesis and nitrate uptake and reduction in chlorophyll content in, Nostoc moscorum, Spirolina plantensis, Anabaena sp., Synechococcus sp. and affect the diversity of microalgal growth in oil polluted areas (23, 24). Microalgae in the oil-polluted environment can tolerate some degree of pollution, but petroleum compounds at high concentrations as due to problems with damage membranous will inhibit their growth (25). It is important to isolate, purify, identify and study oil decomposition microorganisms (bacteria, microalgae and etc.) in rich reserves countries (26). In the present study, a green alga Chlorella sorokiniana was isolated from oil contaminated areas of Khark Island. C. sorokiniana was present in 4 areas around and inside the oil region of lagoon A, oil lagoon C and coastal area southwest of Khark Island. Due to the presence of live algae in water and soils contaminated with petroleum compounds in the area, these strains may have the potential to remove oil contamination and have the ability to break down oil contaminants which it requires further investigation for evaluation.

Based on morphologic investigation three suspected isolates were not identified certainly which two of them belonging to the Pseudanabaenaceae family of cyanobacteria. The first isolate (species Kh.C.d2) of the Khark Island was similar to Anabaenopsis abijatae with cylindrical and elongated cells, while the adjacent genera, Anabaena, and Nostoc cells are spherical. The isolated cyanobacteria similar to A. abijatae have terminal heterocyst however in Anabaena and Nostoc the heterocyst cells are intracellular and interstitial. The heterocyst and akinet in A.abijatae are far with distance but in Anabaena, they are close to each other. However, our isolate has two more differences; firstly the heterocyst diameter of Anabaenopsisis 7 to 10 µm but our isolate is 2 to 4 µm. In addition, A. abijatae has generally spherical akinet and less oval in shape while, on the isolated species from kharak, akint are generally cylindrical and barrels in shape and are less spherical. Also, the akinet diameter of A. abijatae is 12 to 14 µm but in isolate is 6 to 8 µm. Therefore, the species of Anabaenopsis is not clear and need more investigations.

Another isolate (species Kh.T.1) morphologically was similar to Arthronema africanum species with some differences. In A. africanum trichome is 0.8 to 5 µm thick without mucilage cover. There are irregularly swollen cells along the columnar cells, which always occur in Trichome (27), whereas in our case cyanobacteria not only has a distinct gelatinous sheath around the trichome but also no swollen cells were seen. Unlike, some areas of trichome cells have depression. Further molecular tests required to confirm the stains.

The third cyanobacterium (species Kh.T.2) was isolated from Baghe Pire Mard area with polymorph morphology which made it difficult to identify. It is morphologically similar to Pseudanabaena teremula from Pseudanabaenaceae family with simple cylindrical and 4 µm width trichome (seldom 6 µm), cell length is greater than the width, and the cell has a definite border (28). Differences of our isolate with P. teremula is the presence of obvious gelatinous sheath around the trichome, whereas it is absent in P. teremula. Due to polymorphism, some of the cells along the trichome are short and seeming cells are disk-shaped while in P. teremula all trichome cells are cylindrical shape and length is larger than the width (17).

According to the results obtained here, our isolated strains were identified at the genus level based on microscopic morphological examination and it was not possible to identify any isolate

at the species level. The isolated strains appeared morphologically distinguishable from each other ranged from microalgae with a unicellular organization to colonial and filamentous forms with either branched or unbranched organization. Further investigations are directed at identifying any isolate at species level using precise molecular and physiological approaches as well as their possible role in natural biodegradation process of oil.  

 

Acknowledgment

Co-authors would like to fully appreciate of Iranian biological resource center (IBRC) for Cooperation project expenses and laboratory facilities.

(1)  Noroozi M., Naqunezhad A., Mehrvarz Sh S. Algal flora in first Iranian land-marine the Boujagh National Park. International Journal on Algae, DOI: 10.1615/InterJAlgae.v11.i3.70. 2009;11, (3): 276-288.

(2)  Faghir MB., Shafii S. Floristic study on the algae of Siahdarvishan River in Guilan Province, North Iran. Caspian Journal of Environmental Sciences 2013; 11 (1): 111-1266.

(3)  Nejadsattari T., Noroozi M., Fallahi M. The composition and seasonal distribution of epiphytic algae in Anzali lagoon, Iran. Cryptogamie Algologie 2005; 26: 387–398.

(4)  Sohrabipour J., Rabiei R. The checklist of green algae of the Iranian coastal lines of the Persian Gulf and gulf of Oman. Iranian Journal botany 2007; 13 (2):146-149

(5)  Zarei-Darki B. Taxonomic Structure of the Algal Flora of Iran. Bangladesh Journal of Plant Taxonomy 2009; 16(2): 185-194.

(6)  Zarei-Darki B. Marine Species in the Algal Flora of the Anzali Swamp (Iran). Russian Journal of Marine Biology 2009; (35), 3: 200–205.

(7)  Mohamadian E., Karimi N., Rasekh B., Ghasempour H., Dehlavi S. Isolation, Screening and Identification of Diatoms from Kermanshah Oil Refinery Wastewater Treatment Systems. Biological Journal of Microorganism 2014; 11: 47-599.

(8)  Sohrabipour J & Rabiei R The checklist of green algae of the iranian coastal lines of the persian gulf and gulf of oman. The Iranian Journal of Botany 2007; 13 (2) 146-149.

(9)  Carrera-Martnez D., Mateos-Sanz A., Lopez-Rodas V., E. Costas. physiological and genetic response of Dunaliella tertiolecta (Chlorophyceae) to oil samples from the tanker Prestige. Aquatic Toxicology 97 (2010) 151–159. doi:10.1016/j.aquatox.2009.12.016

(10)             Vaziri A., Panahpour E and Mirzaee Beni M.H. Phytoremediation, a Method for Treatment of Petroleum Hydrocarbon Contaminated Soils. International Journal of Farming and Allied Sciences 2013; Vol., 2 (21): 909-913.

(11)             Ehdaei M. Oil pollution and its impacts on aquatic ecosystems and the Persian Gulf. Proceeding of the National Conference of aquaculture and aquatic ecosystems, University of Savadkooh, 1391. http://www.civilica.com/Paper-AQUATIC01-AQUATIC01_025.htm

(12)             Bold HC., Wynne M. Introduction to the Algae. Structure and Reproduction. 2nd ed. Prentice Hall, Inc., Englewood Cliffs, NJ. 706 pp, 1985.

(13)             Chu S P. The influence of the mineral composition of the medium on the growth of planktonic algae: part I, Methods and culture media. The Journal of Ecology 1942: 284-3255.

(14)             Bischoff H W., Bold H C. Phycological studies. IV. Some soil algae from Enchanted Rock and related algal species. University of Texas, Austin, 95 pp, 1963.

(15)             Richmond A. Handbook of microalgal culture: biotechnology and applied phycology. New York: John Wiley & Sons; 2008.

(16)             Whitford LA., Schumacher GJ. Manual of the fresh-water algae in North Carolina. North Carolina Agricultural Experiment Station. Technical Buletten.1969. no. 188.

(17)             Wehr JD., Sheath RG. Freshwater algae of North America: ecology and classification. Elsevier Sciences (USA) Academic Press: San Diego, CA. 950 pp; 2003.

(18)             Desikachary TV. Cyanophyta, Indian Council of Agricultural Research, monographs on Algae. New Delhi, India; 1959.

(19)             Prescott GW. Algae of the western Great Lakes area. Dubuque, Iowa: WC. Brown Company; 1004 pp, 19622.

(20)             Zehnder A., Hughes EO. The antialgal activity of Acti-dione. Canadian journal of microbiology 1958; 4(4): 399-4088.

(21)             Reddy MV., Rao SL., Rao CS. Preliminary study of different media and various process parameters on the growth of Blue-Green Algae (Anabaena ambigua). International Journal of Pharma and Bio Sciences 2013; 4(3): 140- 148

(22)             Domìnguez-Bocanegra AR., Legarreta I., Jeronimo F., Campocosio A. Influence of enviromental and nutritional factors in the production of astaxanthin from Haeamatococcus pluvialisBioresource Technology 2004; 92: 209-21

(23)             Kabli SA. Effect of crude oil and naphthalene on the evolution of oxygen by three species of marine algae. Faculty of Meteorology, Environment and Arid Land Agriculture 1998; 9: 137-1444.

(24)             Sundaram S., Soumya KK. Study of physiological and biochemical alteration in cyanobacteriumunder organic stress. American Journal of Plant Physiology 2011; 6: 1-16.

(25)             Gaur JP., Singh AK. Growth, photosynthesis and nitrogen fixation of Anabaena doliolum exposed to Assam crude extract. Bulletin of environmental contamination and toxicology 1990; 44(3): 494-500.

(26)             Pelletier E., Delille B. "Crude oil bioremediation in sub-Antarctic intertidal sediments: chemistry and toxicity of oil residues". Marine Environmental Research 2004; 57: 311-327.

(27)             Komarek J., Lukavsky J. Arthronema, a new cyanophyte genus from Afro-Asian deserts. ArchivfürHydrobiologie, Supplement. 1988; 80: 249-267.

(28)             Anagnostidis K., Komárek J. Modern approach to the classification system of cyanophytes. 3. Oscillatoriales. Archiv für Hydrobiologie/Algological Studies 1988; 50/53: 327-4722.