آزمون توان ساخت بیوسورفکتانت باکتری جدا شده از یک خاک آلوده به ترکیب های نفتی در کشتگاه‌ها و دماهای گوناگون

نوع مقاله: کاربردی

نویسندگان

1 هیات علمی

2 دانشجو

چکیده

بیوسورفکتانت­ها ترکیبات آمفی­فیلیک هستند که ریزجانداران گوناگونی توانایی ساخت آنها را دارند. این ریزجانداران در صنایع گوناگون و پاکسازی زیستگاه های آبی و خاکی کارایی ویژه ای دارند. این پژوهش با هدف جداسازی باکتری‌های سازنده بیوسورفکتانت انجام شد و توان ساخت بیوسورفکتانت توسط باکتری‌ جداسازی شد و در کشتگاه­های دارای کربن آلی گوناگون )نفت سفید، گلوکز و ملاس نیشکر( در دماهای 30 و 37 درجه سلسیوس و زمان گرماگذاری 48 و 156 ساعت بررسی و ارزیابی گردید. برای شناسایی توان ساخت بیوسورفکتانت از روش‌های کمی و کیفی غربالگری گوناگونی مانند توان همولیز، پراکنش نفت، فروپاشی نفت، فعالیت امولسیون­کنندگی، سنجش آب‌گریزی یاخته و اندازه‌گیری کشش رویین بهره­گیری گردید؛ سپس برپایه یافته­ها بهترین زیستگاه برای ساخت بیوسورفکتانت باکتری بررسی شد. در این پژوهش توان یک جدایه سازنده بیوسورفکتانت در شرایط گوناگون بررسی و آزمون شد. سویه جداسازی شده دارای همولیز مثبت یا β در محیط آگار خون­دار بود. ترکیب­های ساخته شده سویه جداسازی شده در همه کشتگاه­های با کربن آلی گوناگون در زمان‏ها و دماهای به‌کار رفته به ته چاهک ته نشین شدند. این باکتری بیشترین کاهش کشش رویین را در کشتگاه دارای ملاس پس از 48 ساعت و در دمای 30 درجه سلسیوس داشت و توانست کشش رویین آن ‏را تا 83/22 میلی­نیوتون بر متر کاهش دهد؛ همچنین بیشترین درصد امولسیون‏کنندگی (2/56 درصد) نیز در کشتگاه دارای ملاس پس از 48 ساعت و در دمای 30 درجه سلسیوس به­دست آمد. بیشینه درصد آبگریزی رویه یاخته در کشتگاه دارای ملاس (63/66 درصد) دیده شد. برپایه این نتایج نیاز به تحقیقات بیشتری برای استفاده از ملاس نیشکر به عنوان مواد پسماند کشاورزی برای تولید بیوسورفکنانت در سطح تجاری برای کاربردهای مختلف است.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Biosurfactant production ability Assay of a bacterium isolated from oil contaminated soil at different media and temperatures

نویسندگان [English]

  • Naeimeh Enayatizamir 1
  • Shila Khajavi 2
1 Agriculture Faculty
چکیده [English]

Introduction Biosurfactants or microbial surfactants are surface active molecules that are produced from a variety of microorganisms. Due to its amphiphilic nature, these biomolecules are capable of lowering the surface tension, interfacial tension and forming micro-emulsion to enable mixing of two immiscible solutions. Such properties exhibit excellent detergency, emulsifying, foaming and dispersing traits, which can be applied in various industries. The features that make them commercially promising alternatives to chemically synthesized surfactants are their lower toxicity, higher biodegradability, better foaming properties, and greater stability towards temperature and pH. Limited full scale production has been realized for many biosurfactants due to expensive raw material, low production yield and high purification cost. In order to alleviate these problems, many studies have been carried out using cost-free or low-cost feed stocks or agricultural byproducts as substrates for biosurfactant production. Oil pollution and remediation technology has become a global phenomenon of increasing importance.
Materials and Methods In this study, Potential strains of microorganism were isolated from various hydrocarbon polluted area on nutrient agar medium using sterile saline (0.85% NaCl) method and different bacterial isolates were selected based on the colony morphology on nutrient agar. The selected isolates were screened for the production of biosurfactants using following screening methods. Pure culture of bacterial isolates were streaked on the freshly prepared blood agar and incubated at 37°C for 48-72 h. Results were recorded based on the type of clear zone observed i.e. α-hemolysis when the colony was surrounded by greenish zone, β-hemolysis when the colony was surrounded by a clear white zone and γ-hemolysis when there was no change in the medium surrounding the colony. Surface tension reduction and emulsification index of isolates was determined by culturing the isolates in minimal mineral salt solution containing glucose as carbon source. Based on the screening test results, the positive isolates were inoculated into the mineral salt medium for the biosurfactant production and then identified by its microscopic appearance, biochemical tests based on Bergey’s manual of determinative bacteriology and molecular method. Bio-surfactant production by superior isolate was evaluated in minimal mineral salt medium containing different carbon sources (kerosene, sugar cane molasses, phenanthrane and glucose) at 30 and 37 °C within the incubation periods of 48 and 156 hours. Emulsification activity, oil spreading, drop collapse, cell hydrophobicity and surface tension activity of isolate were used to detect biosurfactant production. 
Results and Discussion Out of 13 isolates of microorganism, strain S10 showed positive response to biosurfactant tests (hemolytic activity, surface tension reduction and emulsification index) and was select for identification and considering the effect of different carbon sources on its biosurfactant production. The biochemical and molecular identification results showed isolate S10 belongs to Bacillus pumilus. Results showed that Bacillus pumilus was able to grow in all carbon sources. Based on bio-surfactant production, this strain had a positive or β hemolysis on blood agar medium. Results showed that this bacterium was able to grow in all carbon sources. The compound produced by this strain in each of carbon sources at both temperatures (30 and 37 °C) and incubation periods (48 and 156 hours) collapsed down. The maximum surface tension reduction was recorded in the samples containing molasses as carbon source incubated at 30 ° C for 48 hours, in which bacterium reduced surface tension to 20.33 mNm-1. The highest bacterial growth with a higher surface tension reduction selected this isolate as a potential biosurfactant producing microorganism. The maximum emulsifying and cell hydrophobicity were also recorded in molasses (28%) and kerosene (70%) respectively. 
Conclusion In conclusion, the study represented surfactant activity of the bacterial strain isolated from oil contaminated soils. This confirms that environment has an influence on the metabolism of the tested microbes. This study suggests that, Bacillus pumilus isolated from oil contaminated soil showed biosurfactant producing ability. Further study on the utilization of agro industrial wastes as substrates for the large-scale production of biosurfactants is recommended.

کلیدواژه‌ها [English]

  • Fertilizer
  • Growth
  • phosphorus
  • Potassium
  • Zinc