نوع مقاله : مقاله پژوهشی
نویسندگان
1 گروه زیست فناوری دریا، دانشکده علوم و فنون دریایی، دانشگاه خلیج فارس، بوشهر، ایران
2 گروه محیط زیست، دانشکده مهندسی محیط‌زیست دانشگاه علوم و فنون دریایی خرمشهر
چکیده
هدف از این تحقیق، جداسازی و شناسایی مولکولی باکتری‌های همراه با اسفنج با پتانسیل تجزیه زیستی نفت خام می باشد. بافت مزوهیل اسفنج Pachychalina sp. هموژنیزه شده و رقت های مختلف آن در محیط کشت های مناسب کشت داده شد. کلنی های به دست آمده بر اساس شاخص امولسیون سازی و میزان رشد در محیط حاوی 2% نفت غربالگری شدند. از بین باکتری های جداسازی شده، 6 باکتری بالاترین میزان رشد در محیط نفتی را دارا بودند که میزان حذف نفت توسط 6 سویه مذکور در محیط نمکی حداقل مورد سنجش قرار گرفت. سویه ها همچنین بر اساس توالی ژن SrRNA 16 و با انجام PCR مورد شناسایی مولکولی قرار گرفتند. نتایج حاصل از بررسی درصد حذف نفت بر اساس دو روش جذب در 420 نانومتر و میزان وزن خشک تایید کننده یکدیگر بوده و هر دو از یک الگو پیروی نمودند. بر این اساس، سویه های KE5 و KE8 بیشترین میزان حذف نفت را نشان دادند که نتایج شناسایی مولکولی این دو سویه مشخص نمود بیشترین شباهت را به ترتیب با سویه های Staphylococcus aureus subsp.N315 و Luteimonas terricola BZ92r دارا می باشند. با توجه به نتایج به دست آمده از همترازی های ژنومی، تعیین فاصله ژنتیکی و رسم درخت فیلوژنتیکی، به نظر می رسد سویه های KE6 و KE7 به ترتیب با داشتن 86% و 90% شباهت با سویه های Exiguobacterium sp. AT1b و Pseudomonas rhodesiae CIP 104664 پتانسیل قابل توجهی برای انجام مطالعات بیشتر مولکولی و بیوشیمیایی دارا بودند.
کلیدواژه‌ها
موضوعات
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Isolation and Molecular Identification of Oil Degrading Bacteria Associated with Pachychalina sp. Sponge
Khanomnaz Ebadi[1], Mandana Zarei1 *, Ali Mohammad Sanati3
1. Marine biotechnology, Faculty of Marine Science and Technology, Persian Gulf University, Bushehr, Iran
2. Persian Gulf Institute, Department of environment, Bushehr, Iran
Abstract
The aim of this study was to isolate and molecular identification of associated bacteria in sponges, with potential ability of biodegrading crude oil. Serial dilutions of homogenized Pachychalina sp. mesohyl were cultured in suitable medium for growth of marine microorganism. Obtained colonies were screened based on emulsification index and growth in medium containing 2% oil. Six strains which showed the highest growth rate and emulsification index were tested for the amount of oil removal in the minimal salt medium. Also the molecular identification was done Based on the 16SrRNA sequence and PCR. Removal of oil based on two methods; dry weight and absorption at 420 nm confirmed each other and both followed the same pattern. Accordingly KE5 and KE8 strains showed the highest degree of oil removing and molecular identification results revealed that they were most similar to strains of Staphylococcus aureus subsp. N31 and Luteimonas terricola BZ92r respectively. Also according to the results of bioinformatics analysis, it seems KE6 and KE7 respectively with 84% and 90% similarity with Exiguobacterium sp. AT1b and Pseudomonas rhodesiae CIP 104664 strains, have considerable potential for further molecular and biochemical studies and there is the possibility of introducing them as new strains.
Keywords:Emulsification, Thermophile, Halophile, Persian Gulf, Marine bacteria
Table 1: Materials required for polymerase chain reaction
Table 2: Polymerase Chain Reaction Program
Table 3: Systematic Specifications of crude oil degradation strains
Table 4: The closest strain of the database to the sequence of the 16SrRNA gene of the sequenced bacteria
Table 5: Growth rate of bacteria and oil degradation
Figure 1: Image of the 16srRNA gene electrophoresis for isolated strains
Figure 2: Emulsion index chart for 6 isolates
Figure 3: emulsification index
Figure 4: Growth of KE7 strain at concentrations of 2, 5, 10 and 15% salt
Figure 3: emulsification index
Figure 4: Growth of KE7 strain at concentrations of 2, 5, 10 and 15% salt
Figure 5: The percentage of oil removal based on absorbance at 420 nm
Figure 6: The percentage of crude oil removal based on dry weight
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