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

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

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 4^ºC 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

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

Values is mg/l

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

(+)growth (-)lack of growth

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