Zeynab Bayat1 , Nazanin Akbari2, MehdiHassanshahian1,* , Simone Cappello3, and Ali Salehinasab1
1 Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran
2 Department of Biology, Faculty of Sciences, Shahid Beheshti University, Tehran, Iran
3 Institute for Biological Resources and Marine Biotechnology (IRBIM), CNR of Messina, Italy *
Corresponding author: Mehdi Hassanshahian, Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Email: mshahi@uk.ac.ir
A B S T R A C T
Introduction: Biosurfactants or surface-active compounds with amphiphilic molecular structures, including a hydrophilic and a hydrophobic domain, are produced by microorganisms. These compounds increase the biodegradation of hydrocarbons in
the environment due to their ability to emulsify hydrocarbon-water mixtures. This study was conducted to isolate and characterize biosurfactant-producing bacteria from the samples of Gastropods.
Materials and Methods: The gastropod samples were collected from oilcontaminated sites in the Persian Gulf, Middle East. Biosurfactant-producing strains were isolated from these samples. The biosurfactant production ability was analyzed using Drop Collapse TEST, oil spreading test, emulsification activity test, and BATH test.
Results: In total, 11 biosurfactant-producing strains were isolated. Two isolates with higher growth rates and biosurfactant production ability were selected for further studies. The best isolates were identified as Halomonas sp. isolate BHA16 and Vibrio alginolyticus isolate BHA 17 based on molecular analysis. Gas chromatography analysis of remaining crude oil confirmed that these strains could degrade to 51.44 % and 67.58% of crude oil, respectively.
Conclusion: The results of this study indicated the surfactant activity of the bacterial strains isolated from Gastro pods had a good potential for the biodegradation of crude oil and could be used for the cleanup of oil-contaminated marine environments.
Keywords: Biodegradation, Biosurfactant, Gastropod, Halomonas sp., Vibrio alginolyticus
1. Introduction
The Persian Gulf is an important marine environment in the south of Iran, the pathway for about 60 percent of the marine transported oil in the world. The Persian Gulf was polluted with crude oil during the 1991 Gulf war1. Oil spills in the sea have become a global problem in industrialized and developing countries. It is one of the most dangerous pollution factors known today2. Oily wastewater contains toxic substances, such as phenols, petroleum hydrocarbons, and polyaromatic hydrocarbons, inhibiting plant and animal growth and being mutagenic and carcinogenic to people3,4. It can be a threat to the environment, necessitating monitoring and cleanup of pollution to protect the environment5. One of the strategies to enhance the cleanup is bioremediation which degrades environmental pollution by microorganisms6. Many bacteria from contaminated areas can produce biosurfactants. Biosurfactants produced by hydrocarbondegrading bacteria with amphiphilic molecular structure
(including a hydrophilic and a hydrophobic domain) can be emulsified hydrocarbon-water mixtures, which enables them to grow on the oil droplets and increase the biodegradation of hydrocarbons in the environment7,8.
Several approaches measure the surface activity of biosurfactants, including hemolysis in blood agar, oil spreading, drop collapse test, emulsification activity, and BATH test9-11. Therefore, this study aimed to identify biosurfactant-producing bacteria from gastropod samples collected from oil-contaminated areas in the Persian Gulf. Additionally, the degradation capacity of these strains was examined.
2. Materials and Methods
2.1. Sampling
To isolate biosurfactant-producing bacteria, gastropod samples were collected from oil-contaminated sites in the Persian Gulf in August 2016. This station was located on Qeshm Island, Iran (HA: Haghni Harbor). Gastropods were collected by a knife from a depth range of 5-15 meters. Collected samples were transported on ice to the laboratory within 6 hours. Gastropod samples were washed with marine water twice12. Finally, the solution of gastropods was used for subsequent studies.
2.2. Isolation of biosurfactant-producing bacteria
The ONR7a medium was supplemented with 1% (v/v) of crude-oil (Iranian light crude oil) as the sole carbon source
and energy used to isolate biosurfactant-producing strains of bacteria. ONR7a contained (per liter of distilled water)
40g of NaCl, 11.18 g of MgCl2.6H2O, 3.98 g of Na2SO4, 1.46 g of CaC12.2H2O, 1.3 g of TAPS0 {3-[N tris(hydroxymethyl)
methylamino]-2 hydroxypropanesulfonic acid}, 0.72 g of KCl, 0.27 g of NH4Cl, 89 mg of Na2HP04.7H20, 83 mg of NaBr, 31
mg of NaHCO3, 27mg of H3BO3, 24 mg of SrCl2.6H20, 2.6 mg of NaF and 2 mg of FeCl2.4H20. For solid media, Bacterial
Agar (15 g/l) was added12. All materials were purchased from Merk Co., Germany. A gastropod solution (5 ml) was added to Erlenmeyer flasks containing 100 ml of medium, and the flask was incubated for 7 days at 30°C on a rotary shaker (180 rpm, INFORS AG). Then, 5 ml was transported to a fresh medium. After a series of four subcultures, inoculum from the flask was streaked out, and phenotypically different colonies on ONR7a agar were purified. Phenotypically, different colonies obtained from the plates were transferred to fresh medium with and without crude oil to eliminate autotrophs and agar-utilizing bacteria. The procedure was repeated, and the only isolates exhibiting pronounced growth on crude oil were stored in stock
media with glycerol at -20°C for further characterization13.
