upJ Bioremed Biodegrad
ISSN:2155-6199 JBRBD, an open access journal
Journal of Bioremediation & Biodegradation - Open AccessResearch Article
OPEN ACCESS Freely available online
Volume 1 Issue 21000107
Bioremediation of Crude Oil Contamination Using Microbial Surface-Active Agents: Isolation, Production and CharacterizationArumugam Gnanamani*, Varadharajan Kavitha, Narayanasamy Radhakrishnan and Asit Baran Mandal
Microbiology Division, Central Leather Research Institute, Adyar, Chennai 20, Tamil Nadu, India
*Corresponding author: Dr. A. Gnanamani, Microbiology Division, Central Leather Research Institute, Adyar, Chennai 20, Tamil Nadu, India, Tel: 91-44-24404955;
Fax: 91-44-24912150; E-mail: email@example.com, gnanamani3@
Received September 13, 2010; Accepted October 10, 2010; Published October
Citation: Gnanamani A, Kavitha V, Radhakrishnan N, Mandal AB (2010)
Bioremediation of Crude Oil Contamination Using Microbial Surface-Active Agents: Isolation, Production and Characterization. J Bioremed Biodegrad 1:107. doi:10.4172/2155-6199.1000107
Copyright: 2010 Gnanamani A, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
The present study highlights isolation, screening, production, characterization of marine microbial surface active agents followed by evaluating the effi cacy of said surface -active agents for bioremediation of crude oil contamination. Upon screening, six potential surface-active agents producing isolates of Bacillus genera with signifi cant difference in their morphology were obtained from marine sediments of Tamil Nadu coastal area. Results from optimization studies revealed, sucrose and yeast extract were the suitable carbon and nitrogen sources for growth, pH of 7.2 0.2, temperature of 37C and agitation at 180-200 rpm were the other optimized variables for the maximum production of surface-active agents irrespective of the bacterial isolates. Extraction and characterization studies reveals, the product was polymeric in nature with the surface activity in the range of 28 4 mN/m. Thermal stability was comparable with that of synthetic surfactants and exhibit appreciable emulsifying activity and emulsion stability (more than 90 days). Laboratory scale studies on removal of crude oil from aqueous phase demonstrate, >90% of crude oil was removed
within 60-120 minutes of exposure to the partially purifi ed surface-active agents. The percentage removal showed
signifi cant difference for the six different surface-active agents.
Keywords: Bioremediation; Crude oil removal; Biosurfactant;Marine Bacillus sp. Surface tension; Emulsion
Oil spills and subsequent oil removal is a major global problem. The recent oil spill tragedy in Gulf of Mexico (April 2010) spreads slowly and affects the environmentally sensitive areas too. The sudden huge volume of the oil exposure affects most of the native living populations. Though, both Government and Non- Government agencies initiated necessary steps to safe guard the lives, still, the impact has not yet retrieved completely till this date.
In general, surface tension of water and other liquids are reduced in the presence of synthetic organic chemicals, such as surfactants or wetting agents. Though, use of these agents may solve the above said problem, however, adding these synthetic agents to the site, in amplify the percentage level of non-degradable agents, which, further intensify the problem. These surface-active compounds, in general, contain both hydrophilic and hydrophobic (lipophilic) groups and based on their ionic behaviour in solution they are classified as anionic, cationic, non-ionic or amphoteric surfactants. The hydrophobic part of a surfactant molecule was generally derived from a hydrocarbon containing 8 to 20 carbon atoms (e.g., fatty acids, paraffin, olefins and alkyl benzenes) and the hydrophilic portion may either ionize in aqueous solution (anionic or cationic) or remain non- ionic . Further, recent realization on energy intensive production processes, hazardous nature  and the wastes generated during production restricts, use of synthetic surfactant for clinical and non-clinical applications, which necessitates the need for biocompatible and environmental benign surface-active agents. The high requirement of biosurfactants for food processing, pharmacology or solubilization of oil has been reported by number of authors. Thus, serious attention is being given at global level to have nontoxic, non-hazardous surface-active agents.
Microbial products of few terrestrial and most of the marine microbial species exhibit pronounced surface and emulsifying activities and are classified as bioactive compounds. Compounds that reduce surface tension at air water interface are further grouped as
biosurfactants and those involved in reducing the interfacial tension between immiscible liquids or at the solid- liquid interfaces are called as bioemulsifiers. According to Karanth et al.  biosurfactants usually exhibit emulsifying capacity but bioemulsifiers do not necessarily reduce surface tension. The three major important characteristics of surface-active agents; (i) enrichment at interfaces, (ii) lowering interfacial tension and (iii) micelle formation were also exhibited by the biosurfactants.
Most of the microbial surfactants are lipid in nature and grouped into glycolipids, phospholipids, lipopeptides, natural lipids, fatty acids and lipopolysacharides . Nevertheless, the origin and the strain types decide the nature and the surface-active property of biosurfactants. According to Maneerat  microorganisms of marine origin exhibit the maximum yield and surface-active property compared to terrestrial species. Numbers of reports are available on glucose lipids, trehalose lipids from marine Alcanivorax borkumensis and Arthrobacter sp. ornithine lipids from marine Myroides sp. SM1, polymeric biosurfactants from marine Yarrowia lipolytica and Pseudomonas nautical and rhamnolipid from marine Pseudomonas aeruginosa A41. In addition, according to Abdul and Gibson  and Bai et al.  removal of oil/hydrocarbons from contaminated soil using commercial biosurfactants in its purified form, showing better results than that of chemical surfactants such as sodium dodecyl
Citation: Gnanamani A, Kavitha V, Radhakrishnan N, Mandal AB (2010) Bioremediation of Crude Oil Contamination Using Microbial Surface-Active Agents: Isolation, Production and Characterization. J Bioremed Biodegrad 1:107. doi:10.4172/2155-6199.1000107
lishing GroupJ Bioremed Biodegrad
ISSN:2155-6199 JBRBD, an open access journal Volume 1 Issue 21000107
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sulphate (SDS) and Tween 80. Similarly Urum et al.  reported management of crude oil in sand using commercial biosurfactants showed excellent results. However, a significant barrier to the widespread use of biosurfactant in environmental applications is the recovery of product from the production media. Santa Anna et al.  and Santos et al.  produced a crude fermented medium from Pseudomonas aeruginosa PA1 containing a blend of five types of RML, which utilized a low-cost raw material (glycerol). The use of crude biosurfactants, a viable option, limits with the research finding on the nature of biosurfactants and the organisms used for the production.
In the present study, we aimed to remove oil from aqueous phase using microbial surface-active agents. In detail, the study describes, isolation, production (optimization of nutrients and environmental factors) and characterization of surface-active agents from marine microbes and the efficacy of the said surface-active agents for the removal of oil from aqueous phase.
Materials and Methods
Collection and isolation
Marine samples of water, sediments, mussels, shells and sand were collected from Kalpakkam, Ennore port, Besant Nagar Beach, Marina beach, Mahabalipuram beach, Mandapam, Vedaranyam, Tuticorin, Cuddalore in Tamil Nadu, India, according to the procedures summarized by Saravanan et al. .
Sabourauds dextrose agar (for fungi) and Zobell marine broth and agar (for bacteria) were the media used for the isolation of microbial species according to the standard procedure employed for the cultivation and maintenance of marine organisms. Fungi species were identified through observation of hyphal growth on agar plates and microscopic observation of cell dimensions. Morphologically distinct microbial colonies, screened and classified based on their Grams reaction. All the pure cultures are stored at - 80C in the presence of 30% of glycerol.
Screening of biosurfactants producing microorganism
For screening of biosurfactants producing organisms, all the obtained pure cultures were grown in Zobell medium individually at
37C under 150 rpm for 48 h and the biosurfactants activity of the cell free medium was assessed according to the methods of Tugrul
and Cansunar , and the isolates exhibiting appreciable surfactant activity was selected and examined for identification and further
Identification of selected isolates by phenotypical and molecular level assessment
Followed by screening, the selected isolates (six numbers) were subjected to phenotypical, biochemical and molecular assessment for identification. Gram staining followed by microscopical examination and all the req